JPH07143900A - Polynucleotide for use in amplifying single primer and oligonucleotide containing phospho- thioate as primer in amplifying nucleic acid. - Google Patents

Abstract

A method is disclosed for extending an extender probe to produce a single stranded polydeoxynucleotide that is free of unreacted extender probe and has two segments that are non-contiguous and complementary with each other. The method comprises the steps of (1) providing in combination (a) a polynucleotide having two non-contiguous, non-complementary nucleotide sequences S1 and S2 wherein S2 is 5' of S1 and is at least ten deoxynucleotides long, (b) an extender probe comprised of two deoxynucleotide sequences, wherein the sequence at the 3'-end of the extender probe (EP1) is hybridizable with S1 and the other of the deoxynucleotide sequences (EP2) is substantially identical to S2 and (c) means for modifying the 3'-end of extender probe that does not hybridize with the polynucleotide and (2) extending the extender probe along the polynucleotide wherein extender probe not hybridized to the polynucleotide becomes modified at its 3'-end. A method is also disclosed for forming a polynucleotide sequence complementary to a single stranded target polynucleotide sequence ("target sequence"). In the method a polynucleotide primer having a 3'-terminus comprised of a nucleotide monophosphate, which has at least one phosphate-sulfur bond, is hybridized to the 3'-end of the target sequence. The polynucleotide primer is extended along the target sequence and the extended polynucleotide primer is then dissociated from the target sequence. The method finds particular application in the area of nucleic acid amplification.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION Nucleic acid hybridization has been used for its identification and proof of its existence. Hybridization is based on the binding of complementary base pairs. When complementary single-stranded nucleic acids are incubated together, the complementary base sequences pair to form a double-stranded hybrid molecule. Single-stranded deoxyribonucleic acid (ssDN
The ability of A) or ribonucleic acid (RNA) to form hydrogen bonding structures with complementary nucleic acid sequences has been used as an analytical tool in the study of molecular biology. The availability of highly specific radioactive nucleoside triphosphates and the ability of the T4 kinase to label DNA with ³² P allowed the identification, isolation and characterization of various nucleic acid sequences of biological interest. Nucleic acid hybridization
It has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences result from genetic or environmental changes in the DNA due to insertions, deletions, point mutations, or the acquisition of foreign DNA or RNA by bacteria, filamentous fungi, fungi and viruses. To date, nucleic acid sequences have been used primarily in molecular biology laboratories in universities and industry. Nucleic acid hybridization as a diagnostic tool in clinical medicine due to the very low concentration of disease-related DNA or RNA in the body fluids of patients and the lack of sufficiently sensitive methods for nucleic acid hybridization analysis. Has limited applications.

Current methods for detecting specific nucleic acid sequences use the immobilization of target nucleic acids on solid supports such as nitrocellulose paper, cellulose paper, diazotized paper, or nylon membranes. After the target nucleic acid is immobilized on the support, the support is contacted with the appropriately labeled probe nucleic acid for about 2 to 48 hours. After this time, the solid support is washed several times at a controlled temperature to remove unhybridized probe. The support is then dried and the hybridized material is detected by autoradiography or spectroscopic methods.

If it is necessary to detect very low concentrations,
Current methods are time consuming and laborious, and unlike radiolabels, non-radioactive labels that are not readily available are often not suitable. Therefore, methods that increase sensitivity to allow the use of simple, rapid, non-radioactive, homogeneous or ratio homogenous methods for detecting nucleic acid sequences are preferred.

Recently, polymerase chain reaction (PCR)
A method of enzymatic amplification of a specific portion of DNA, known as This in vitro amplification method is based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension with a thermophilic polymerase to exponentially increase the copy number of the region between the primers. A PCR primer that anneals to the opposite strand of DNA is positioned such that the polymerase-catalyzed extension product of one primer acts as a template strand for the other primer, the length of which is the 5'end of the oligonucleotide primer. Discontinuous fragments, defined by the distance between them, accumulate.

Another method has recently been described for amplifying nucleic acid sequences. This method is referred to as a single primer amplification method because PCR uses only one primer, unlike PCR which requires two primers. This method provides amplification of target nucleic acids having a stem-loop or inverted repeat base sequence structure flanked by relatively short complementary sequences to the target nucleic acid. Various methods for making such target nucleic acids related to the presence of the polynucleotide to be detected have also been described.

One problem with using synthetic oligonucleotide primers is that such primers are
It is susceptible to degradation by the exonuclease activity associated with many DNA polymerases used in amplification methods. As mentioned above, one such method is PCR, which provides exponential amplification of nucleic acids. PCR is generally performed using Taq polymerase, which lacks 3'-5 'exonuclease activity and is unable to remove mismatches. Many variations of this method have been described, some require cycling of temperature and some use a single temperature. The single temperature method relies on the activity of as many as four enzymes to amplify, and the one using temperature cycling relies on a thermostable polymerase. Genetic polymorphisms or point mutations are commonly reported in the detection of genetic disorders using PCR. Primers mismatched at the 3'end are used to artificially introduce the product or to detect single base mismatches in the target DNA. However, the enzymes containing the 3'-exonuclease activity used in the amplification experimental design digest the mismatched primer-template region, which greatly limits this type of analysis.

[0007] Part of the present invention allows extension of a primer along with a template and amplification of the primer even in the presence of an enzyme having a strong 3'-exonuclease activity.

[0008]

DESCRIPTION OF THE PRIOR ART Methods for amplifying, detecting and / or cloning nucleic acid sequences are described in US Pat. No. 4,683,1.
95 or 4.683,202.
Sequence polymerisation is described by Saiki et al. (1986) S.
Science, 230: 1350-1354. Methods for making oligonucleotides are described in European Patent Application No. 0194545A2.
Belgian Patent Application No. BE904402 discloses templates for making DNA detection probes. Gene amplification in eukaryotic cells is described in US Pat. No. 4,656,134.

Langer et al., Proc.
Natl. Acad. Sci. USA (1981) 7
8: 6633-6637 discloses the enzymatic synthesis of biotin labeled polynucleotides and their use as novel nucleic acid affinity probes. Detection of viral genomes in cultured cells and paraffin-fixed tissues using biotin is described by Brigati et al., Virolog.
y, (1983) 136: 32-50. U.S. Pat. No. 4,486,539 discloses the detection of microbial nucleic acid by a one-step sandwich hybridization test. Sensitive detection of malignant tumors based on DNA detection is described in US Pat. No. 4,490,472. U.S. Pat. No. 4,480,040
Disclosed is a highly sensitive and rapid diagnostic method for a plant viroid disease or virus, which uses a radiolabeled DNA complementary to a nucleic acid of a viroid or virus to be detected. European Patent Application No. 83106112.2 (Priority US Patent Application No. 391,440, filed June 23, 1982) describes modified labeled nucleotides and polynucleotides and their methods of preparation, use and detection. ing. Methods and compositions for detecting and measuring cellular DNA are disclosed in US Pat. No. 4,423,153. Specific DNA probes in diagnostic microbiology are
It is described in U.S. Pat. No. 4,358,535. Methods for detecting polymorphic restriction sites and nucleic acid sequences are described in European Patent Application No. 0164054A1. US Pat. No. 4,663,283 describes a method of altering double-stranded DNA.

Genomic amplification with transformant sequencing is described by Stoflet et al., Science.
e (198) 239: 491. Primer-directed enzymatic amplification of DNA using a thermostable DNA polymerase is described by Saiki et al., Science.
e (1988), 239: 487. US Pat. No. 4,724,202 discloses the use of non-hybridizing nucleic acids for the detection of nucleic acid hybridization. Bugawan et al. Describe the use of non-radioactive oligonucleotide probes to analyze enzymatically amplified DNA for parentage discrimination and forensic HLA typing.

Detection and isolation of homologous, repetitive and amplified nucleic acid sequences is described in US Pat. No. 4,675,2.
No. 83. U.S. Pat. No. 4,683,19
Nos. 5 and 4,683,202 describe a homogeneous polynucleotide displacement assay by digestion of displaced RNA single-stranded polynucleotides from a reagent complex, and separate complementary complementation of nucleic acids with two oligonucleotide primers. Disclosed is the amplification of nucleic acid sequences by treatment of the strands. European Patent Application No. 0200362 describes a method for amplifying, detecting or cloning nucleic acid sequences, which is useful for diagnosing diseases and preparing transformation vectors. A convenient assay for relative nucleic acid levels in a variety of small samples by cytoplasmic dot hybridization is described in US Pat. No. 4,677,054. A method for detecting hybridization of a nucleic acid sequence with a probe containing a thionucleotide is disclosed in US Pat. No. 4,647,529.
No.

A simple and efficient enzymatic method for covalent binding of DNA to cellulose and its application to hybridization-restriction analysis and in vitro synthesis of DNA probes are described in Nucleic Acids Research.
h (1986) 14: 9171-9191. Cleavage of single-stranded oligonucleotides by EcoRI is Nucleic Acids Research
(1987) 15: 709-716.

Exponential amplification of recombinant RNA hybridization probes is described by Lizardi et al.
(1988) Bio / Technology 6:11.
97-1202. Farlander (F
ahrlander) et al. describe amplification of DNA probe signals: A ChristmasTr
ee Approach in Bio / Techno
logy (1988) 6: 1165-1168.

A nucleic acid hybridization assay using a probe capable of cross-linking the target sequence is described in US Pat. No. 4,599,303. This method involves the preparation of specific ribonucleic acid or deoxyribonucleic acid molecules in which a bifunctional cross-linking molecule has been covalently incorporated. This incorporation is such that the cross-linking molecule retains the ability to undergo a second reaction with nucleic acid of a bacterial, viral or mammalian chromosome that is the target of the probe to form a covalent cross-link. . After cross-linking, the non-cross-linked probe is separated from the covalent cross-linked probe-target complex using one of the methods that distinguishes single-stranded probes from double-stranded covalent cross-linked probe-target complexes.

Hybridization methods and probes for detecting nucleic acid sequences are described in US Pat. No. 4,908,30.
No. 7. The amplified hybridization method is described in US Pat. No. 4,882,269, in which a first hybridizing target sequence of interest is used.
A second probe that produces a group of signals binds to the primer.

A method for the detection of target sequences of nucleic acids by hybridization with diagnostic and flanking probes for the diagnosis of genetically abnormal diseases (in particular an automated method) is described in European patent application 0185494A2. . In this method, the sample is hybridized with a probe (diagnostic probe) complementary to the diagnostic part of the target sequence, and the diagnostic part is substantially bound only to the sample nucleic acid containing the target sequence. A probe (adjacent probe) complementary to the nucleotide sequence adjacent to is hybridized. The diagnostic probe and the adjacent probe are then covalently linked to create a target probe that is complementary to the target sequence and the unbound probe is removed. In the preferred method,
One of the probes is labeled with 2 of the sample nucleic acid target probes.
The presence or absence of the target sequence can be determined by lysing the single strands, eluting the dissociated target probe, and testing the label.

The above method has a drawback in that at least flanking sequences are required. To perform this method, diagnostic and flanking sequences must be identified and diagnostic and flanking probes complementary to these sequences must be made. If the diagnostic and flanking sequences are not accurately identified,
The diagnostic probe and the adjacent probe do not hybridize well and the specificity and sensitivity of the assay is lost or substantially reduced.

The DNA amplification and elimination method is W089 / 1269
No. 5 is described. This method uses the isolation of genomic or RNA derived double stranded fragments that are unique to one of the two fragment mixtures. Fragments in the positive and negative source mixtures have separate terminal linkers attached, and each mixture is amplified by continuous replication of the prime strand using a single-stranded primer homologous to the attached linkers. Second
Of the source linkers are biotinylated and the fragments in this mixture are hybridized in molar excess to the fragments in the positive source mixture. Species that do not react with biotinylated species (ie, species unique to the positive source mixture) are isolated after removal of hybridized species by affinity chromatography. Also disclosed is a method of amplifying a mixture of DNA fragments by repeated replication of linkers / primers.

US Pat. Nos. 07 / 299,282 and 07 / 399,795 (January 19, 1989, respectively)
, And filed Aug. 29, 1989) (see also European Patent 379369) describe nucleic acid amplification using a single polynucleotide primer. US patent application Ser. No. 07 / 555,323 (filed Jul. 19, 1990) (see also EP 469755) discloses a method of producing a polynucleotide for use in single primer amplification. The disclosures of these applications are incorporated herein by reference.

DNA and RNA sequence determinations based on phosphorothioate chemistry are described by G. Gish et al.
Science (1988) 240: 1520-152.
2. Hybridization methods for detecting nucleic acids using probes containing thionucleotides are described in US Pat. No. 4,647,529. PCR-based site-directed mutagenesis using primers with mismatched 3'ends is described by M. Nassal et al., Nucleic Acids.
Research (1990) 18 (10): 307
7-3078. G. Sar
kar) et al. (Analytical Biochemi
(Stry (1990) 186: 64-68) discloses characterization of amplification of specific alleles by the polymerase chain reaction. B- for the diagnosis of sickle cell anemia
Allele-specific enzymatic amplification of globin genomic DNA is described in Wu et al., Proc. Natl. Acad. Sc
i. USA (1989) 86: 2757-2760. Modification of enzymatically amplified DNA for the detection of point mutations is described in Haliasso.
s) et al., Nucleic Acids Research
h (1989) 17: 3606. Kwok et al. Describe the effect of primer-template mismatch on the polymerase chain reaction: Nucleic Acids Research.
h (1990) : Human Immunodeficiency Virus Type 1 Model Study in 999-1005. The technical aspects of HLA-DP allele typing and single-base mismatch detection using allele-specific DNA in vitro amplification and sequence-specific oligonucleotide probes are described in Fuger (L. Fugg).
er) et al., J. Immunol. Methods (19
90) 129: 175-185. J. Ott et al., Biochemistry (19
87) 26: 8237-8241 describes the protection of oligonucleotide primers against degradation by DNA polymerase I. The double-stranded stability of the phosphorothioate, methylphosphonate, and RNA analogs of the two DNA 14-mers is due to the Kibler-Herzog (Kible
r-Herzok) et al., Nucleic Acids
Research (1991) 19 (No. 11): 2
979-2981. The effect of phosphorothioate homo-oligodeoxynucleotides on herpes simplex type 2 inducible DNA polymerase was
Gao et al., J. Biol. Chem. (19
89) 262 (No. 19): 11521-11526.
And they observed inhibition of HSV-inducible DNA polymerase and certain human DNA polymerases by sulfur-containing oligonucleotides. Griep et al., Biochemistry (199)
0) 29: 9006-9014 disclose reduction of 3'to 5'exonuclease activity of DNA polymerase III holoenzyme by template-primer analogs.

Phosphoramidite
Tetraethyl thiuram disulfide as a reagent for phosphorothioate oligonucleotide synthesis via midite chemistry
The use of disulfide (TETD) is based on Applied Biosystems (Applied Biosystems).
Ems) has published several publications: 1) Research News, "DNA Synthesis Tetraethylthiuram Disulfide: A New Reagent for Antisense Phosphorothioate DNA" (Research News, "DNA Sy").
nthesis Tetraethylthiouram
Disulfide: A New Reagent f
or Antisense Phosphorothi
oate DNA ”), 2) User Bulletin 58
Issue, February 1991, Models 380A, 380B, 3
81A, 391, 392, 394 DNA synthesizer (Sy
nthesizes) and 3) TETD / acetonitrile propaganda note, No. 401147 "TETD: A.
sissense Phosphorothioate
Synthesis Made Easy ".

Amplification, detection and / or cloning methods for nucleic acid sequences are described in US Pat. No. 4,683,195, 4,
683,202, 4,800,159, 4,96
5,188 and 5,008,182. The sequence polymer method by the polymerase chain reaction is described by Saiki et al. (1986) Science.
e, 230: 1350-1354. Primer-directed enzymatic amplification of DNA using a thermostable DNA polymerase is described in Saiki et al., Scie.
nce (1988) 239: 487. .

Amplification of nucleic acid sequences using random sequence oligonucleotides as primers is described in US Pat.
043,272. The use of phosphorothioate primers in PCR amplification has been used in the freshly published Nucleic Acid Research.
h, Vol. 20, No. 14,3551-3554,
1992.

[0024]

SUMMARY OF THE INVENTION In one embodiment of the present invention, an extension probe and a single-stranded target polynucleotide sequence are the same as the above target polynucleotide sequence without an unmodified extension probe at its 3'end, Provided is a method of making a single-stranded polynucleotide sequence linked to a polynucleotide sequence complementary to the polynucleotide sequence at the 5'end of the single-stranded target polynucleotide sequence. This method comprises the steps of (a) adding the 5'of the target polynucleotide sequence to the 3'end of the single-stranded target polynucleotide sequence.
Hybridizing the 3'end of an extender probe containing a sequence substantially identical to sequence S2 at the end, (b)
This extension probe is extended along the single-stranded target polynucleotide sequence, (c) the 3'end of the extension probe that does not hybridize to the single-stranded target polynucleotide sequence is modified, and (d) the sequence S2 is added to the 3'end. Comprising hybridizing a primer having a to the 3'end of the extended extension probe, and (e) extending this primer along the extended extension probe.

The present invention disclosed herein is a single-chain polydeoxy which has two segments which are not adjacent to each other and which are not adjacent to each other and extend the extension probe modified at its 3'end. It includes methods and reagents for making nucleotides. This method is applied to, for example, a single primer amplification measurement method.

In one embodiment of the tree invention, the extension probe is extended to create a single-stranded polydeoxynucleotide having two non-contiguous two complementary segments. This production method has (a) two non-contiguous non-complementary nucleotide sequences S1 and S2,
S2 is a polynucleotide 5'of S1 at least 10 nucleotides in length, (b) is an extension probe consisting of two deoxynucleotide sequences, and the sequence (EP1) at the 3'end of the extension probe is hybridizable with S1. And the other deoxynucleotide sequence (EP
2) is an extension probe that is substantially identical to S2, and (c) means for modifying the 3'end of the extension probe that does not hybridize to the polynucleotide, and (d) extending the extension probe along the polynucleotide ( In this case the extension probe which does not hybridize to the polynucleotide is modified at its 3'end and comprises a combination of steps.

In the above embodiment of the present invention, (a)
The extension probe is extended along the polynucleotide
The 3'end of the extension probe that forms a double strand and does not hybridize with the polynucleotide (b) is modified, (c) the extended extension probe is dissociated from the double strand, and (d) the polydeoxynucleotide primer is extended. A second 2 which hybridizes with the extended extender and is extended along the extended extender to include the extended primer.
Forming a double strand, (e) the extension primer is dissociated from the second double strand, and (f) the primer hybridizes with the extension primer and is extended along the extension primer to include the extension primer. A polydeoxynucleotide primer, a DNA polymerase, and a deoxynucleoside 3 which form a chain and are hybridizable at least at the 3'end with a nucleotide sequence complementary to S2 under conditions in which steps (e) and (f) are repeated. Phosphoric acid is provided in combination.

Another embodiment of the invention is a method for making a single-stranded polynucleotide sequence complementary to a single-stranded target polynucleotide sequence. This method comprises (a) a single-stranded target polynucleotide sequence in a medium, a DNA polymerase activity having 3'-exonuclease activity, and a sequence complementary to the 3'-terminal sequence of the single-stranded target polynucleotide sequence. And (b) treating said medium to combine said extension probe with said single probe. Hybridize to the strand target polynucleotide,
Extending the extender probe along the single-stranded target polynucleotide sequence and degrading the 3'end of the extender probe.

In another embodiment, the presence of the target polynucleotide sequence in the medium suspected of containing the target polynucleotide sequence is detected. This target polynucleotide sequence consists of two non-adjacent non-hybridizable nucleotide sequences, S1 and S2, where S2 is the 5'of S1.
And is at least 10 nucleotides in length). In this method, (a) (1) medium, (2) the 3′-end sequence (EP1) of the extension probe is hybridizable with S1, and another deoxynucleotide sequence (EP2) is used.
Is an extension probe having two deoxynucleotide sequences that are substantially identical to S2, (3) means for modifying the 3'end of the extension probe that does not hybridize to the target polynucleotide sequence, and (4) the primer in situ. A polynucleotide primer capable of hybridizing to a nucleotide sequence complementary to S2 on condition that it is produced,
(5) DNA polymerase and (6) a combination of deoxynucleoside triphosphates simultaneously or in whole or in part in succession, the (A) extension probe hybridizes to the target polynucleotide sequence and the target polynucleotide sequence. An extension probe that is extended along with (extended EP) to form a duplex and (B) does not hybridize to the target nucleotide sequence is modified at its 3'end,
(C) The extended EP is dissociated from the above double strand,
(D) the primer hybridizes to and extends along the extended probe to form a second double strand consisting of the extended primer, (E) the extended primer is dissociated from the double strand, and (F) the primer hybridizes to and extends along the extension primer to form a duplex consisting of the extension primer, steps (E) and (F) providing under repeated conditions, and (b) It consists of checking for the presence of the extension primer.

Another embodiment of the invention relates to a method for detecting the presence of a polynucleotide in a sample suspected of containing the polynucleotide. This method involves (a) treating a medium containing a sample from the polynucleotide (if present) of two non-contiguous non-complementary nucleotide sequences S1 and S2, where S2 is the 5'of S1. And is at least 10 nucleotides in length), and (b) in the medium (1) at the 3'end of the extension probe (EP).
1) is hybridizable with S1 and the other deoxynucleotide sequence (EP2) is substantially identical to S2,
An extension probe having two deoxynucleotide sequences, (2) a nucleotide sequence (NS) having a portion capable of hybridizing with EP1, which is another molecule or portion of the extension probe, (3) polydeoxynucleotide A polydeoxynucleotide primer capable of hybridizing to a nucleotide sequence complementary to S2, (4) deoxynucleoside triphosphate, (5) and DNA if there is no means to degrade the extended probe to form the primer Template-dependent polydeoxynucleotide polymerase (A) extension probe
An extension probe that hybridizes to the target polynucleotide sequence and is extended along the target polynucleotide sequence (extended extension probe) to form a double strand and (B) does not hybridize to the target polynucleotide sequence is NS. And (C) the extended extension probe is dissociated from the double strand, and (D) the primer hybridizes with and is extended along with the extended extension probe to comprise the extended primer. Forming a double strand, (E) the extension primer is dissociated from the double strand, and (F) the primer hybridizes with and is extended along to form a double strand consisting of the extension primer, the steps of: (E) and (F) under repeated conditions (wherein steps (a) and (b) are carried out simultaneously or in whole or in part) The connection effected), in combination, and (c) a method comprising the step of determining the presence of the extension primer.

Another embodiment of the present invention relates to a method for detecting the presence of a polynucleotide in a sample suspected of containing the polynucleotide. This method involves (a) treating a medium containing a sample from the polynucleotide (if present) of two non-contiguous non-complementary nucleotide sequences S1 and S2, where S2 is the 5'of S1. And is at least 10 nucleotides in length), and (b) in the medium (1) at the 3'end of the extension probe (EP).
1) is hybridizable with S1 and the other deoxynucleotide sequence (EP2) is substantially identical to S2,
An extension probe having two deoxynucleotide sequences, (2) an enzyme capable of degrading a single-stranded polynucleotide, (3) no means for degrading the extension probe to form a polydeoxynucleotide primer, A polydeoxynucleotide primer capable of hybridizing with a nucleotide sequence complementary to S2, (4) deoxynucleoside triphosphate, (5) and a DNA template-dependent polydeoxynucleotide polymerase (A) an extension probe targeting An extension probe that hybridizes to the polynucleotide sequence and extends along the target polynucleotide sequence to form a duplex, (B) an extension probe that does not hybridize to the target polynucleotide sequence is degraded and (C) extended. The extension probe is 2 above.
Dissociated from the double strand, (D) the primer hybridizes with and is extended along with the extended extension probe, (E) the extended primer is dissociated from the double strand, and (F) the primer is extended primer. Hybridizes and is extended along it to form a double strand consisting of an extension primer, wherein steps (E) and (F) are repeated under repeated conditions (steps (a) and (b) being performed simultaneously or in whole). Or partially sequentially), in combination, and (c) determining the presence of the extension primer.

The present invention further comprises (a) a sequence (EP1) at its 3'end which is hybridizable with the first sequence in the target polynucleotide sequence and is substantially the same as the second sequence of the target polynucleotide sequence. A polydeoxynucleotide extender probe having the sequence (EP2) identical to (wherein the second sequence in the target polynucleotide sequence is 5'of the first sequence and is not adjacent to the first sequence), (b)
Means for modifying the 3'end of the extender probe that does not hybridize to the target polynucleotide sequence, and (c) a second
And a polydeoxynucleotide primer capable of hybridizing to a sequence complementary to the sequence of SEQ.

1-8 are schematic diagrams of different embodiments of the present invention.

[0034]

DESCRIPTION OF SPECIFIC EMBODIMENTS The present invention relates to single-stranded (ss)
Extending an extender probe along the target polynucleotide sequence to allow production of a single-stranded polynucleotide with the ability to form an intramolecular base-paired structure (where it is not involved in the production of the single-stranded polynucleotide) The 3'end of the extension probe is modified). Thus, a single-stranded polynucleotide has an intramolecular base-pairing structure, ie two non-adjacent mutually complementary segments (inverted repeats (invert repeats)).
ed repeat). This method has particular application in the field of amplification of single-stranded primers as described above, where the target polynucleotide sequence in the sample has inverted repeats or can be converted to such a structure, if the target polynucleotide sequence is The nucleotide sequence is amplified. The present invention minimizes the number of reagents and steps required,
It provides an extremely convenient method for converting to a target polynucleotide sequence having an intramolecular base pairing structure.

The present invention, in a broad sense, provides for the production of a target polynucleotide sequence having inverted repeats formed from the extension probe, where all extension probes that do not hybridize to the target polynucleotide sequence are 3 '.
Modified at the ends and thus not present in unmodified form in the medium containing the newly formed single-stranded polynucleotide). The target polynucleotide sequence is combined with an extender probe in the medium, wherein the extender probe is (1) the 3'end of the extender probe complementary to the first sequence at the 3'end of the target polynucleotide sequence within the target polynucleotide sequence. And (2) a second sequence of an extension probe that is substantially identical to the second sequence of the target polynucleotide sequence, wherein each second sequence is 5'of the first sequence. . The medium is treated to hybridize the extender probe to the target polynucleotide sequence, the extender probe is extended along the target polynucleotide sequence, degrading the 3'end of the extender probe that does not hybridize to the target polynucleotide sequence.

Before further describing the specific embodiments of the present invention, some terms are defined.

Polynucleotide--a compound or composition to be measured, which is a polymeric nucleotide,
This is about 20 to 500 in the intact natural state,
Can have 000 or more nucleotides, can have about 30 to 50,000 or more nucleotides in the isolated state, usually about 100 to 20,000, and more often Is 500
To 10,000 nucleotides. It is therefore clear that fragmentation occurs when isolated from its native state. A polynucleotide comprises a purified or unpurified form of nucleic acid from any source,
NA (dsDNA and ssDNA) and RNA (t-RN
A, m-RNA and r-RNA), mitochondrial DNA and RNA, chloroplast DNA and RNA, DNA-RN
A hybrid, or a mixture thereof, a chromosome, a plasmid, a genome of a biomaterial (for example, a bacterium such as a bacterium, a yeast, a virus, a viroid, a filamentous fungus, a heart muscle, a plant, an animal, or a human), a fragment thereof, or the like. Polynucleotides are only a small part of a complex mixture such as a biological sample. It is obtained from various biological samples by methods known to those skilled in the art. An example of such a biological sample is shown in Table 1 and is for illustration only and in no way limits the invention.

[0038]

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

The polynucleotides are treated and cleaved, eg, by shearing, or endonucleases or other site-specific chemical cleavage methods to yield fragments containing the target polynucleotide sequence. However, it is an advantage of the present invention that the polynucleotide can be used in isolated form without further cleavage.

For the purposes of the present invention, a polynucleotide or a cleaved fragment obtained from a polynucleotide is treated to be at least partially denatured or single-stranded, or denatured or single-stranded. It Such treatments are known to those skilled in the art and include, for example, heat treatment or alkali treatment. For example, double-stranded DNA is 90-
It produces a denatured material when heated at 100 ° C. for about 1 to 10 minutes.

Target Polynucleotide Sequence--A sequence of nucleotides to be identified, usually present within a polynucleotide, the body of which is a preparation of an extended probe polydeoxynucleotide which hybridizes to at least a portion of such target sequence. Is known to the extent that it allows at least 10 nucleotide segments at its 3'end, and preferably 15, often 20 to 5
It is a 0 nucleotide segment, which comprises at least a 10 nucleotide segment that is substantially identical at its 5'end. The target polynucleotide sequence comprises two non-contiguous non-complementary nucleotide sequences (S1 and S2), one of which (S1) is a moiety capable of hybridizing to the extended probe polydeoxynucleotide and S2 is a 5 '). The target polynucleotide sequence usually contains about 30 to 5,000 or more nucleotides, preferably 50 to 1,000.
Contains 0 nucleotides. The two non-contiguous non-complementary nucleotide sequences (S1 and S2) are preferably each 1
It has 0 to 100 nucleotides and is separated by at least 10 bases, preferably 100, usually 200 to 10,000 bases. One target polynucleotide sequence is often part of a polynucleotide. The target polynucleotide sequence is generally a fraction of a larger substance, or is substantially the entire molecule. The minimum number of nucleotides in the target polynucleotide sequence is selected to ensure that the presence of the target polynucleotide sequence in the sample is a specific indicator of the presence of the polynucleotide in the sample. Very roughly speaking, the length of the sequence is usually greater than about 1.61 og L nucleotides, where L is the number of base pairs in the genome of the biological source of the sample. The maximum number of nucleotides in a target sequence is usually determined by the length of the polynucleotide and its shearability, or by any method required to prepare the sample for other manipulations and measurements during isolation. , And the efficiency of detection and / or the efficiency of amplification of the sequence.

Single-stranded polydeoxynucleotide sequence--
A sequence of deoxynucleotides formed as a result of the present invention. It usually consists of at least two non-adjacent complementary segments or flanking sequences. It is also a specific binding site for a receptor, such as a repressor, restriction enzyme, which when bound to its complementary strand, contains one or more sequences. The first or second segment or flanking sequences are respectively at the 3'and 5'ends of the single stranded polynucleotide sequence and each is at least 10, preferably at least 1
It consists of 5 deoxynucleotides and / or its derivatives.

Single-stranded polydeoxynucleotide sequences usually contain 30 to 10,000 deoxynucleotides, preferably 100 to 2,000 deoxynucleotides, more preferably 500 to 5,000 nucleotides. When a single-stranded polydeoxynucleotide sequence is hybridized with complementary strands, each end is a member of a pair of inverted repeats.

Polydeoxynucleotide primer--
A synthetic deoxynucleotide, usually a single strand,
A polydeoxynucleotide having a sequence identical to the sequence S2, or a sequence capable of hybridizing with a nucleotide sequence complementary to the sequence S2 of the target polynucleotide sequence, at its 3 ′ end. Usually the polydeoxynucleotide primer has at least 90%, preferably 100%, the same base sequence as the second nucleotide sequence EP2 of the extension probe. The number of deoxynucleotides in the hybridizable sequence of the polydeoxynucleotide primer depends on the stringency used to hybridize the polydeoxynucleotide primer.
The cy) conditions must be such as to prevent excess non-specific hybridization. Usually, the number of deoxynucleotides in the polydeoxynucleotide primer will be at least S2 of the target polynucleotide sequence.
As large as in the sequence, ie at least 10 deoxynucleotides, preferably at least 15 deoxynucleotides, generally about 10 to 200, preferably 20 to 50 deoxynucleotides.

Deoxynucleoside triphosphate-5'3
A deoxynucleoside having a phosphoric acid substituent. Deoxynucleosides are pentose derivatives in which a purine or pyrimidine derivative of the nitrogen-containing base is covalently attached to the 1'-carbon of the pentose. Purine bases include adenine (A), guanine (G), inosine, and their derivatives and analogs. Pyrimidine bases include cytosine (C), thymine (T), uracil (U), and their derivatives and analogs.

Derivatives and analogs are exemplified by those that are recognized and polymerized in a manner similar to underivatized nucleoside triphosphates. Examples of such derivatives or analogs include those modified with reporter groups, those that are biotinylated, those that are amine modified, those that are radiolabeled, those that are alkylated, etc. Phosphites, ring atom modified derivatives and the like are included, but these are for illustrative purposes only and are not meant to limit the invention in any way. The reporter group is
It may be a fluorescent group such as fluorescein, a chemiluminescent group such as luminol, and a terbium chelating substance such as N- (hydroxyethyl) ethylenediaminetriacetic acid that can be detected by delayed fluorescence.

Polydeoxynucleotide polymerase-
A catalyst, usually an enzyme, for making an extended strand of a polydeoxynucleotide primer along a DNA template in which the extended strand contains a complementary single-stranded polydeoxynucleotide. Polydeoxynucleotide Polymerase A template-dependent polydeoxynucleotide polymerase that uses deoxynucleoside triphosphates as a building block to extend the 3'end of polydeoxynucleotide primers. Usually the catalyst is an enzyme, for example a prokaryotic DNA polymerase (I, II
Or III), T4 DNA polymerase, T7 DNA
DNA for polymerase, Klenow fragment, reverse transcriptase, etc.
There are polymerases, which are derived from cells, bacteria (eg E. coli), plants, animals, viruses, thermophilic bacteria and the like. The polynucleotide or target polynucleotide sequence is RNA
, A reverse transcriptase will be included to facilitate extension of the extension probe along the polynucleotide or target polynucleotide sequence.

In whole or in part continuously--when the sample or various agents used in the present invention are combined, not simultaneously, one or more agents may be combined with the remaining one or more agents. Combined with the drug to make one combination.
Each combination is then exposed to one or more steps of the present invention. That is, each combination is incubated under conditions that achieve one or more desired results.

Hybridization and binding--the terms are interchangeable in the sense of nucleotide sequences. The ability of two nucleotide sequences to hybridize to each other is based on the degree of complementarity of the two nucleotide sequences, which is also based on the percent match of complementary nucleotide pairs. The more nucleotides that are complementary to one sequence in another, the more stringent the conditions of hybridization and the more specific the binding of the two sequences. The stringency is increased by increasing the temperature, increasing the ratio of cosolvents and decreasing the salt concentration.

Homologous or Substantially Identical--Two polynucleotide sequences that are generally identical or that hybridize to the same polynucleotide sequence are homologous. Two
Two sequences are homologous or substantially identical if at least 90%, preferably 100%, have identical or similar base sequences (wherein thymine (T) and uracil (U) are identical). I think). Thus ribonucleotides A, U, C and G are considered similar to deoxynucleotides dA, dT, dC and dG, respectively. Both homologous sequences may be DNA,
One may be DNA and the other RNA.

Complementary--One of the two sequences is anti-parallel to the other (the 3'end of each sequence binds to the 5'end of the other sequence and each A, T (U) of one sequence, Two sequences are complementary when G and C bind at T (U), A, C and G of the other sequence, respectively.

Extended probe--This is a single-stranded polynucleotide chain, usually a synthetic oligonucleotide, consisting of two nucleotide sequences, one of such sequences located at the 3'end of the chain (EP1). ) Preferably has at least 10 consecutive deoxynucleotides and is capable of hybridizing to the first polynucleotide sequence (S1) of the target polynucleotide sequence.

The major criterion for choosing EP1 is (1) the array must be reliable, that is, S
It should be almost or completely complementary to 1 and of sufficient length to provide stable and specific binding.
(2) The N-terminus must have or be capable of forming a free 3'-hydroxy group. The minimum length of EP1 is usually at least 10, usually at least 15, preferably 20-50 deoxynucleotides. Generally EP1 is about 20 to 100 deoxynucleotides. The combined length of the first and second polynucleotide sequences of the extender probe is at least about 2
It is 0 nucleotides, preferably about 40 to 200 nucleotides in length.

The second polynucleotide sequence (EP2) of the extender probe is a nucleotide sequence which is substantially identical or homologous to the second polynucleotide sequence (S2) of the target polynucleotide sequence. EP2 is at least 10 nucleotides, usually at least 15, preferably 2
The length is 0-50 deoxynucleotides. Generally E
P2 is about 20 to 100 deoxynucleotides.

The extender probe contains additional receptor binding or spacer sequences or other sequences located between EP1 and EP2 or at the end of EP2.

Non-contiguous--The sequences are non-contiguous means that there is usually at least 10 nucleotides present in the target polynucleotide sequence between the two segments of the polynucleotide or between the two sequences S1 and S2. means.

Adjacent--A sequence is considered contiguous if no nucleotides are present between the two segments of the polynucleotide or between the two sequences.

Copy--refers to a direct, identical or homologous copy of such a single-stranded polynucleotide, distinguished from sequences which are complementary to the sequence of the single-stranded polynucleotide. In the single primer amplification carried out in the present invention, the complementary sequence of the single-stranded polydeoxynucleotide sequence is first produced as a result of the extension of the polydeoxynucleotide primer and then the direct sequence of the single-stranded polydeoxynucleotide sequence. A sequence that is a copy of the above is obtained from the complementary sequence.

Means for Extending the Extender Probe--The extender probe having an extendable 3'end is an extender probe hybridized to a polynucleotide such as a target polynucleotide sequence under conditions for extending the extender probe,
It can be extended in combination with polydeoxynucleotide polymerase and deoxynucleoside triphosphate. Thus, the extension probe is extended along the polynucleotide to form a double strand. When extended along the target polynucleotide sequence, the duplex consists of an extended probe. Extension by this method provides the required accuracy between the two sequences and the extended extension probe provides accurate detection of the target of interest.

Means for modifying the 3'end of the extender probe-
-For the single primer amplification described above, a single polynucleotide chain capable of forming a base loop structure or inverted repeats is used. Such a polynucleotide is present in the sample or is made in response to the presence of the polynucleotide. An extension probe is used to make such a polynucleotide using binding to a target polynucleotide along which the extension probe is extended. Since the concentration of polynucleotides is generally low and unknown, some extender probe molecules do not hybridize to the target polynucleotide sequence. Molecules of these extension probes are not preferred because of the competing process that reduces the efficiency of single primer amplification. Using suitable means, the 3'end of the extension probe that does not bind to the target polynucleotide sequence is modified so that it will no longer extend along the target polynucleotide sequence in the presence of deoxynucleoside triphosphate or DNA polymerase. become.

One way in which the 3'end of the extension probe is modified is by degradation. An enzyme such as 3'-exonuclease is added to the reaction medium. Under certain conditions such enzymes degrade the 3'end of single-stranded polynucleotides. Examples of such exonuclease enzymes include, but are not limited to, Klenow fragment, T4 polymerase, and T7 polymerase. In one method, the polydeoxynucleotide polymerase, such as the DNA polymerase used to extend the extender probe, has exonuclease activity. Examples of such DNA polymerases are Klenow, T4 and T7 DNA polymerases.

In another embodiment, the 3'end of the extender probe is extended along a scavenger polynucleotide having the sequence NS except at its 5'end, which sequence hybridizes to the 3'end of the extender probe. Soybean is possible. When the 3'end of the extender probe is hybridized to the scavenger polynucleotide sequence, the 3'end of the extender probe is extended along the scavenger polynucleotide sequence in the presence of polydeoxynucleotide polymerase and deoxynucleoside triphosphate. This method modifies the 3'end of the extender probe, eliminating the extension of the extender probe along the target polynucleotide sequence or complementarity during single primer amplification. The scavenger polynucleotide sequence is generally 8 to 1,000 or more nucleotides, preferably 10 to 50 nucleotides in length, which is either a part of the extender probe or a different molecule from the extender probe. When the scavenger polynucleotide sequence is part of an extender probe, it may be 3'or 5'of the extender probe sequence EP2. The enzyme used in this chain extension is a commercially available thermophilic nucleotide polymerase such as, but not limited to, Taq, Vent, Hot Tub and the like.

A member of a specific binding pair ("sbp member")-specifically binds to its surface or cavity within a particular spatial or polar structure of another molecule and is therefore defined as complementary. One of two different molecules with regions. The members of the specific binding pair are called ligand and receptor (anti-receptor). These are members of immunological pairs such as antigen-antibody, or operator-repressor, nuclease-nucleotide, biotin-
Avidin, hormone-hormone receptor, nucleic acid double chain, IgG-protein A, DNA-DNA, DNA-RNA
And so on.

Ligand--any compound for which a receptor naturally exists or is prepared.

Receptor (“antiligand”)-any compound capable of recognizing a particular spatial and polar structure of a molecule (eg, an epitope or antigenic determinant site). Examples of receptors include naturally occurring receptors such as thyroxine binding globulin, antibodies, enzymes, protective enzymes, protein A, complement component Clq, DNA binding proteins or ligands.

Small organic molecules--compounds of molecular weight less than 1500, preferably 100 to 1000, more preferably 300 to 600 (eg biotin, fluorescein, rhodamine and other dyes, tetracycline and other protein binding molecules, and haptens). . Small organic molecules provide a means for attachment of the nucleotide sequence to a label or support.

Support or Surface--A porous or non-porous water-insoluble material. The support may be hydrophilic or made hydrophilic, inorganic powders such as silica, magnesium sulphate and alumina; natural polymeric materials, especially cellulosic and cellulosic derived materials (eg filter paper, chromatography). Fibers containing paper such as paper); synthetic or modified naturally occurring polymers (eg nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross-linked dextran,
Agarose, polyacrylate, polyethylene, polypropylene, poly (4-methylbutane), polystyrene,
Polymethacrylate, poly (ethylene terephthalate), nylon, poly (vinyl butyrate), etc.);
Used by itself or with other substances; Glass available as Bioglass,
There are ceramics and metals.

The attachment of the sbp member to the support or surface is usually carried out by methods known in the literature. For example, "Immobilized Enzymes," Ichiro Chibatra, Halted Press New York (1978), and Cuatrcass.
ecasas), J. Biol. Chem. , 245:
3059 (1970). The shape of the surface may be any of the following (eg strips, rods, particles such as beads, etc.).

Label or reporter group or reporter molecule--a member of the signal producing system. A common label or reporter group or molecule is attached to or becomes attached to the polynucleotide probe or polydeoxynucleotide primer, which can be detected directly or through a specific reaction and is a detectable signal. To produce. Labels include amplification or ligat
ion) or a polynucleotide primer or specific polynucleotide sequence that acts as a ligand such as a repressor protein. Preferably the polydeoxynucleotide primer becomes or can carry a label. Generally any detectable label is used. Labels are radioactive or non-radioactive (usually non-radioactive) and include catalysts (eg enzymes), polynucleotides encoding the catalysts, promoters, dyes, fluorescent molecules, luminescent molecules, coenzymes, enzyme substrates, radioactive groups, There are small organic molecules, amplifiable polynucleotide sequences, particles such as latex or carbon, metal sols, crystallites, liposomes, cells, etc., which may be further labeled with catalysts or other detectable groups. The label is a member of the signal producing system and produces a detectable signal alone or together with other members of the signal producing system. The label is attached directly to the nucleotide sequence or is bound thereto by an sbp member complementary to the sbp member attached to the nucleotide sequence.

Signal Production System--The signal production system contains one or more components, at least one component being a label or reporter group. The signal producing system produces a signal related to the presence or amount of the target polynucleotide sequence or polynucleotide in the sample. The signal producing system contains all the reagents necessary to produce a measurable signal. When the label is not bound to the nucleotide sequence, the label normally binds to an sbp member that is complementary to the sbp member bound to the nucleotide sequence or a portion thereof. Other components of the signal producing system are contained in the developing solution and include substrates, enhancers, cofactors, inhibitors, scavengers, metal ions, and specific binding substances required for binding the signal producing system.
Other components of the signal producing system include coenzymes, substances that react with enzyme products, and other enzymes and catalysts. The signal producing system produces a signal that is detectable by external means, electromagnetic radiation, preferably by visual inspection. This signal producing system is described in more detail in European Patent No. 469755 (US patent application Ser. No. 07 / 555,323, filed Jul. 19, 1990), the related disclosure content of which is hereby incorporated by reference. It is quoted in the book.

Adjuncts--A variety of adjuncts are used in the measurements in connection with the present invention. For example, there is usually a buffer in the measuring medium, and there are stabilizers and measuring components of the measuring medium. Often in addition to these adjuncts, proteins such as albumin, organic solvents such as formamide, quaternary ammonium salts, polycations such as dextran sulfate,
Included are surfactants (especially non-ionic surfactants), binding enhancers (eg polyalkyl glycols) and the like.

In one aspect of the invention, the extender probe is extended along the target polynucleotide sequence and the extender probe that does not hybridize to the target polynucleotide sequence is modified at its 3'end. Methods for making a single-stranded polynucleotide sequence complementary to a nucleotide sequence are provided.

One embodiment of this method is shown schematically in FIG. E located at the 3'end of the extension probe
P1 hybridizes to S1 of the target polynucleotide sequence and to a portion of the scavenger polynucleotide containing the sequence NS. EP2 is homologous to S2. This extension probe is extended along A and S2
Produces an extended extender probe containing the sequence S'2 complementary to. B includes EP2 and S'2, which are hybridizable to each other. The extension probe that does not hybridize to the target polynucleotide sequence hybridizes to the NS and is extended along the scavenger polynucleotide to produce modified extension probe C, where EP1 is no longer present at the 3'end. Preferably the NS is about 8 to 100, more preferably 8 to 30 nucleotides in length.

Another embodiment of the present invention is shown in FIG. In this embodiment, the extension probes are EP1 and EP.
Not only 2 but also the sequence NS, which is the 3'end of the sequence EP2. NS is homologous to S1. EP1 located at the 3'end of the extender probe is the S of the target polynucleotide sequence.
1 and hybridizes with NS of the extension probe. EP2 is homologous to S2. This extension probe extends along A and is complementary to S2 and has the sequence S '.
Produces an extended probe D containing 2. D
Contains EP2 and S'2, which are hybridizable to each other. The extension probe, which does not hybridize to the target polynucleotide sequence, spins and hybridizes to itself, and EP1 hybridizes to NS. The 3'end of the extension probe is extended along itself to produce a modified extension probe E, here EP
1 is no longer present at the 3'end.

Another embodiment is shown in FIG. 3 in which the sequence NS is contained in the extension probe 5'of EP2. Hybridization and extension of the extension probe with the target polynucleotide sequence and itself occurs as described in Figure 2 above.

In yet another embodiment of the invention described in FIG. 4, the extender probe EP1 hybridizes to S1 of the target polynucleotide sequence. The extender probe is extended along A to produce extender probe B extended as described in the embodiment of FIG. An exonuclease with 3'activity was present in the reaction mixture that reduced the extension probe that did not hybridize to A to a sufficient extent to destroy the ability of the extension probe to hybridize with S1 at its 3'end. To achieve.

A variation of the embodiment of FIG. 4 is shown in FIG. This extension probe is not only for EP1 and EP2, but for E
It also includes EP3, which is a sequence hybridizable to P2 and preferably complementary to at least the 3'end of EP2.
EP1 of this extension probe hybridizes to S1 of the target polynucleotide sequence. The extended extension probe B is formed as described in the embodiment of FIG. 4 above. Exonuclease degrades the extended probe, which does not bind to the target polynucleotide sequence, to the double strand formed by hybridizing EP3 and EP2. The extension probe of this embodiment is at least E due to its degradation.
Designed to have P1 removed. Preferably E
P2 is about 5 to 50, more preferably 8 to 30 nucleotides in length. This variant provides the option of using the extended probe degraded in the following steps as a primer, where the EP of the degraded extended probe is
2 binds to S2 of the extended extension probe and is extended along the extended extension probe.

This method is used for single primer amplification in which one or more target polynucleotide sequences (ie, sequences identical to the target polynucleotide sequence) without extension probes are formed. The extender probe is hybridized to the target polynucleotide sequence and extended as described above. The extension probe that does not bind the target is modified at its 3'end in any of the above embodiments. Then the polydeoxynucleotide primer
(1) The extended extension probe is made into a single strand, (2)
The polydeoxynucleotide primer hybridizes with the extended extension probe and is extended along the extended extension probe to form a duplex consisting of the extension primer, which contains the same sequence as the target polynucleotide sequence. Under conditions that hybridize to a nucleotide sequence complementary to S2 at least at its 3'end. Preferably the concentration of extender probe is substantially lower than the concentration of polydeoxynucleotide primer. By "substantially low" is meant that the concentration of extender probe to primer is at least 1: 10, usually 1: 100 or more. Preferably the concentration of extender probe is less than 1% of the concentration of polydeoxynucleotide primer.

The use of the method of the invention for single primer amplification is described in FIG.

[0080] polydeoxynucleotide primer P has its 3 'end S'2 sequences hybridizing to ^{(S M}
₂ ) and S′2 is S2 of the target polynucleotide sequence.
Is complementary to. Preferably S ^M 2 has the same sequence as S2. P may also contain a label W. P hybridizes with and is extended along with the extended extension probe B (FIG. 1), D (FIG. 2) or F (FIG. 3) to provide an extension primer H consisting of S ^M 2 and S ^{M ′} 2. formed, S ^{M '2} is complementary to EP2 preferably S'2
Is the same as B, D or F and H are dissociated and P
Is S ^{M '2} and B, D or S'2 hybridize F of H, P is producing B, and each H and ^{H 1} is extended along a D or F and H. H ¹ has a sequence complementary to S′2 and S ^M 2. The duplex is dissociated, hybridized with H ¹ and H and extended along them to produce H ¹ and H ² . Repeating this further yields multiple copies of H ¹ H ² , which are detected due to the presence of label W.

In one embodiment of the invention, the method is used to modify the 3'end of an extension probe to form a polydeoxynucleotide primer in situ.
This embodiment is described in FIG. The extension probe contains EP1 and EP2, and EP2 is the primer sequence S ^M
It is equivalent to 2 and contains the labeled substance W at any time. The extender probe EP1 hybridizes to S1 of the target polynucleotide sequence and a sequence (NS3) in the scavenger polynucleotide, which is complementary to at least a portion of EP2. S2 is homologous to EP2. The extender probe extends along A and is of the sequence S'2 which is complementary to S2.
To produce an extended probe B containing B is now EP2 and S'which are hybridizable to each other.
Contains 2. The extension probe hybridized to NS3 is degraded by an exonuclease with 3'activity, which is added to the reaction medium. The extension probe is made so that its degradation produces the polydeoxynucleotide primer P, which is used in a single primer amplification. Therefore, NS3 hybridizes to EP2 at least at its 3'end, and EP1 is degraded by exonuclease to yield 3'of the remaining polynucleotide.
EP2 remains at the end. NS hybridized to EP2
Due to the presence of the double strand formed by 3, the reaction conditions are chosen such that no further degradation occurs.

Another convenient way to control the degradation of the extension probe to generate polydeoxynucleotide primers in situ is set forth in FIG. This replaces the 3'-exonuclease and 1 instead of the phosphate diester between the last and penultimate nucleotides of EP2.
One or more phosphorothioate diesters (-
S) (shown in FIG. 8 by S). Degradation of the extended probe that does not bind to the target polynucleotide sequence stops at the phosphorothioate diester or at one or more nucleotides 3'of the phosphorothioate. The degraded extension probe has the sequence EP2 at the 3'end with phosphorothioates near the 3'end and acts as primer P to extend the chain in a single primer amplification. In this embodiment EP2 and S ^M2 are the same.

When the present invention is applied to the replication of a target polynucleotide sequence, one of the above embodiments is carried out and the following steps are repeated at least once: (a) an extended extension of the polydeoxynucleotide primer. Hybridizing with and extending along the probe to form a second duplex of extended probes,
(B) The extension primer is dissociated from the second double strand.
Usually, this method is repeated at least three times, where the primer also hybridizes with and extends along with the extension primer to form a duplex consisting of the extension primer and the extension primer is dissociated. Preferably at least the 15 nucleotide sequence EP1 of the extension probe is S
Hybridizes with 1. Preferably also, the polydeoxynucleotide primer contains at least 15 deoxynucleotide sequences S ^M 2 capable of hybridizing with a sequence complementary to S2.

Preferably S1 and S2 each contain 10 to 100 nucleotides. The method can also be applied when the target polynucleotide sequence is DNA or RNA. 1
In one aspect the polydeoxynucleotide primer is labeled with a reporter molecule. Reporter molecules may be, for example, detectable or binding groups (eg biotin or S2
Is a nucleotide sequence other than the sequence that hybridizes with the sequence complementary to. The extension primer is detected using the reporter molecule covalently attached to the probe. The probe typically has a nucleotide sequence that is homologous or complementary to a portion of the target nucleotide sequence other than S1 or S2.

Another embodiment of the invention relates to a method of detecting the presence of a polynucleotide in a sample suspected of containing the polynucleotide. The medium containing the sample is treated as described above to form the single-stranded target polynucleotide sequence that may be present from the polynucleotide. The target polynucleotide sequence has two non-contiguous non-complementary nucleotide sequences S1 and S2, where S2 is 5'of S1 and is at least 10 nucleotides in length. The vehicle is combined with an extender probe having two deoxynucleotide sequences. The sequence at the 3'end of the extension probe (EP1) hybridizes with S1. The other deoxynucleotide sequence (EP2) is homologous to S2. Included is a method of modifying the 3'end of an extender probe that does not hybridize to the target nucleotide sequence. If modification of the extension probe does not provide a primer, a polydeoxynucleotide primer capable of hybridizing to the nucleotide sequence complementary to S2 is included. Deoxynucleoside triphosphates and one or more polydeoxynucleotide polymerases are also combined. The following conditions are selected: (1) The extension probe hybridizes with the target polynucleotide sequence and is extended along the same to form a double strand, and (2) the extension probe that does not hybridize to the target polynucleotide sequence is Qualified,
(3) The extended extension probe is dissociated from the double strand,
(4) The primer hybridizes with the extension sequence and is extended along the extension sequence to form a second double strand consisting of the extension primer, (5) the extension primer is dissociated from the double strand, and (6) the primer Hybridizes with the extension primer and is extended along it to form a duplex consisting of the extension primer. Steps (5) and (6) are repeated and steps (a) and (b) are performed simultaneously or wholly or partially sequentially. Next, the presence of the extension primer is examined. The presence of the extension primer indicates the presence of the polynucleotide. Steps (5) and (6) are repeated at least 3 times, preferably at least 10 times; usually less than 30 times. Generally, steps (5) and (6) are repeated a sufficient number of times to accurately detect the polynucleotide. When the polynucleotide is RNA, the polydeoxynucleotide polymerase comprises reverse transcriptase.

In carrying out the method of forming single-stranded polydeoxynucleotides with the extender probe and modifying and amplifying the extender probe that does not hybridize to the target polynucleotide sequence, an aqueous medium is used. Other polar cosolvents are also used, usually 1-6, more commonly 1
-Oxygenated organic solvents of 4 carbon atoms (eg alcohols, ethers etc.) are used. Usually, these cosolvents are present in less than about 70% by weight, or more usually less than about 30% by weight.

The pH of the medium is usually in the range of about 4.5 to 9.5, more usually in the range of about 5.5-8.5,
And it is preferably in the range of 6-8. Simultaneous or sequential dissociation of internally hybridized sequences, extender probe and target polynucleotide sequence and 3 ′ of extender probe
Hybridization with any other sequence that forms part of the means for modifying the ends, hybridization of a polydeoxynucleotide primer with an extended probe and an extended primer, reduction of the 3'end of the extended probe with an exonuclease. So that dissociation of the extended probe and extended primer
Depending on the case, pH and temperature are selected and changed. In one example, the speed of these steps depends on whether it is preferable to perform the steps sequentially or simultaneously.
A compromise is made to optimize efficiency and specificity. Various buffers are used to achieve the desired pH and maintain the pH during the measurement. Examples of buffers include borate buffer, phosphate buffer, carbonate buffer, Tris buffer, barbital buffer and the like. Although the particular buffer used is not critical to the invention, there are preferred buffers for each method.

Moderate temperatures are used in carrying out the method. In practicing the method, the medium is usually cycled at two to three temperatures. The temperature of the process is generally about 10 to 10
5 ° C, more usually about 40 to 99 ° C, preferably 50 to 98 ° C. The exact temperature depends on salt concentration, pH,
It will vary depending on the solvent used, the target and the length of the S1 or S2 sequence and the composition of the target polynucleotide sequence or primer. Relatively low temperatures of about 30 to 65 ° C are used for the extension step, and denaturation and hybridization is about 50%.
To 105 ° C. Degradation of the 3'end of the extension probe with exonuclease is usually performed at a temperature of about 15 to 105 ° C, preferably 20 to 50 ° C.

The time to modify the 3'end of the extension probe that does not hybridize to the target polynucleotide sequence is
Generally about 0.5 to 30 minutes, preferably 1 to 20 minutes. When the method of the invention is used in single primer amplification, it is carried out for a time sufficient to achieve the required copy number of the extended probe or its complementary sequence. It also depends on the purpose for which the amplification is carried out (eg the measurement of polynucleotides). Generally, the time to perform the method is 1
Approximately 1 to 10 minutes per cycle, 1 to 200 times or more, usually 5 to 80 times, often 10-60
One cycle is used. For convenience, it is generally desirable to reduce the time and number of cycles. Generally, some amplification time can be shortened, for example, by choosing a concentration of nucleoside triphosphate sufficient to saturate the polynucleotide polymerase and increasing the concentration of the polynucleotide polymerase or polydeoxynucleotide primer. it can. The time for carrying out the method is generally about 5 to 200 minutes. In terms of convenience, it is usually preferable to shorten the time.

The above conditions are also selected to form the target polynucleotide sequence from the polynucleotide.

The amount of reagent that modifies the 3'end of the extender probe will vary depending on the particular means to accomplish the modification. Where the modification involves extension of the 3'end of the extension probe,
The concentration of template-dependent polynucleotide polymerase and deoxynucleoside triphosphates are generally equal to or higher than the concentrations described in the amplifications below, and require different enzymes. The concentration of reagents used to extend or amplify the extension probe along the target polynucleotide sequence is sufficient to extend the extension probe that does not bind to the target polynucleotide sequence. The concentration of any scavenger polynucleotide sequence is generally at least the same as, or usually at least 10 times higher than, the concentration of the extension probe.

When modifying the extension probe with a 3'-exonuclease, the concentration of the exonuclease is selected to degrade the extension probe to the required extent in a practical amount of time (eg 0.5-20 minutes). It Preferably the template dependent polynucleotide polymerase is also 3'-
The concentration of the polymerase is chosen to have exonuclease activity and thus sufficient to achieve chain extension and degradation. Normally, if the 3'end of the extension probe is completely degraded, the magnesium ion concentration during the initial enzymatic reaction is kept low (eg less than 4 mM) and the pH is kept high (eg 8.0 or higher).

As mentioned above, the concentration of the extension probe is substantially lower than the concentration of the primer. Preferably the concentration of the extension probe is less than 1% of the concentration of the primer, more preferably less than 0.1% of the concentration of the lymer, usually the concentration of the extension probe is less than 1 nanomolar, often 0.1 nanomolar (nM ), The concentration of the primer is usually 10 nanomolar or more, and usually at least 100
It is nanomolar. Preferably the primer concentration is 100
Greater than nM and the concentration of extension probe is less than 1 nM.

The amount of target polynucleotide sequence to be copied is as low as 1 or 2 molecules in the sample, but is generally about 10 ² to 10 ¹⁰ , more usually about 10 ^{3 in the} sample.
To 10 ⁸ molecules, preferably at least 1 in the sample
^0-21 M, ^10-10 to ^10-19 M, and more typically ^10-14 to ^10-19 M. The amount of polydeoxynucleotide primer is at least as high as the required copy number, usually 10 ⁻¹³ to 10 ⁻⁸ mol per sample, and the sample is 1-1000 μl. Ordinarily primers are at least 10 ^-9 M, preferably 10 ^-7 M, more preferably at least about 10 ^-6 M.
It is M. Preferably, the concentration of polydeoxynucleotide primer is substantially in excess of that of the single-stranded polynucleotide, preferably at least a 100-fold excess.

The concentration of deoxynucleoside triphosphates in the medium varies widely: preferably these reagents are present in excess. Ordinary deoxynucleoside triphosphate is 10
^-6 to 10-2M, preferably ^10-5 to ^10-3
It is M.

The concentration of template-dependent polynucleotide polymerase is usually empirically determined. A sufficient concentration is preferably used, and even if the concentration is increased, the time required for amplification is 5
It does not increase more than twice, preferably more than twice. The major limiting factor is generally the cost of reagents.

The order in which the various reagents are combined to create the combination varies. In general, the target polynucleotide sequence will be obtained from a sample containing such a sequence or polynucleotide and treated to obtain such a sequence. Generally, the target polynucleotide sequence and the extension probe are any polynucleotide sequence required for modification of the 3'end of the extension probe that does not bind to the target polynucleotide sequence, deoxynucleoside triphosphates, template-dependent polynucleotide polymerase and optionally 3 '. -Combined with a pre-made combination of exonucleases.
Included in the prepared combination, if necessary,
Or added later. However, all of the above simultaneous additions and stepwise or continuous additions are used.

Reagent concentrations, order of addition and method conditions are generally governed by the copy number of the extended probe, the rate at which such copies are made and the need to increase the fidelity of replication. Generally it is preferred to increase the copy number of the extender probe by at least 10 ² fold, preferably 10 ⁴ fold, more preferably 10 ⁶ fold.

In practicing the method of the present invention as applied to the detection of polynucleotides, media, pH, temperature, and time are considered above.

The concentrations of the various reagents are generally determined by the concentration range of the polynucleotide of interest, but the final concentration of each reagent is usually empirically determined to optimize the sensitivity of the assay in the range of interest. The concentration of other reagents in the measurement is
It is determined according to the same principle as described in the amplification method above.
The main consideration is that a sufficient copy number of the extension primer is produced in the absence of the extension probe as compared to the polynucleotide sequence, and thus such a copy is easily detected, allowing accurate measurement of the polynucleotide. Is to be.

The copy of the extension primer can be detected in various ways. For example, in the method, a molecule of polydeoxynucleotide primer is labeled with a reporter molecule (eg, ligand, small organic molecule, polynucleotide sequence, protein, support, operator-repressor pair, intercalating dye, etc.).

Examples of specific labels or reporter molecules and their detection are given in US patent application Ser. No. 07 / 555,323.
No. (filed Jul. 19, 1990) (see also EP 469755), the relevant disclosure content of which is incorporated herein by reference.

Other methods of measurement and detection are described in US patent application Ser. Nos. 07 / 299,282 (filed Jan. 19, 1989) and 07 / 399,795 (Aug. 2, 1989).
Filed 9th) (see also EP 379369), which are hereby incorporated by reference.

Standard methods of specifically detecting nucleic acid sequences can be used.

One method of detecting nucleic acids uses nucleic acid probes.

One method of using a probe is US Patent Application No. 773,386 (filed Sep. 6, 1985).
U.S. Pat. No. 4,868,104), the disclosure content of which is incorporated herein by reference.

The method of detecting a signal depends on the nature of the signal producing system used. If the reporter group label is an enzyme, additional members of the signal producing system include an enzyme substrate. The product of the enzymatic reaction is preferably a luminescent product, or a fluorescent or non-fluorescent dye (both detected by spectroscopic methods), or other spectroscopically or electromagnetically detected product. It is a thing. When the labeling substance is a fluorescent molecule, the medium is irradiated with light and the fluorescence is measured.
If the label is a radioactive group, the medium is counted to measure the radiation count.

A variety of methods can be used to prepare the extension probes, polydeoxynucleotide primers, or other polynucleotide sequences used in the present invention. These are obtained by biological or chemical synthesis. For short sequences (up to about 100 nucleotides):
Chemical synthesis is often more economical than biological synthesis. In addition to economic concerns, chemical synthetic methods are convenient methods for incorporating low molecular weight compounds and / or incorporating modified bases during the synthetic process. In addition, chemical synthesis methods are very flexible in choosing the length and region of the target polynucleotide binding sequence. Extension probes, polydeoxynucleotide primers and other polynucleotides can be synthesized using standard methods (eg, commercially available automated nucleic acid synthesizers). Chemical synthesis of DNA on appropriately modified glass or resin yields surface bound DNA. This is advantageous for cleaning and sample handling. Standard replication methods used in molecular biology are used for longer sequences (eg J. Messaging, (1983) Me.
methods Enzymol. 101, 20-78 for the use of M13 on single-stranded DNA).

Other methods of oligonucleotide synthesis include:
The phosphotriester and phosphodiester method (Narang et al., (1979) Meth. Ez.
ymol 68:90) and synthesis on a support (Beaucage et al., (1981) Tetr.
ahedron Letters 22: 1859-1
862), and the phosphoramidate method, Caruthers, M., et al., "Methods i.
n Enzymology, "Vol. 154, 287.
-314 (1988), and "Synthesis."
and Applications of DNA a
nd RNA, "Narang.
Hen, Academic Press (Academic Press)
s), New York, 1987, and in the literature included herein.

Extender probes containing at least one phosphorothioate diester are prepared by known methods. Oligonucleotide synthesis is carried out as described above up to the point where the introduction of phosphorothioate diesters is preferred. The phosphorothioate diesters are introduced in a variety of ways (eg, oxidation with commercially available dicyclosulfide or thiolation reagents such as tetraethylthiuram disulfide). The remaining nucleotides are then introduced. Other methods for preparing phosphorothioates containing polynucleotides are described in W09908838) W08.
911486, U.S. Pat. No. 4,910,300, European Patent 318245, the relevant disclosures of which are incorporated herein by reference. Other methods of preparing phosphorothioate containing polynucleotides are described in (a) Yau et al., Tetra.
hedron Lett. (1990) 31 (14):
1953-1956; (b) Brill et al.,
Ibid. (1989) 30 (48): 6621-6624;
(C) Caruthers et al., Nucl
eic Acids Symp. Ser. (1989)
21: 119-120; (d) caruturs (carut)
hers) et al., Nucleosides Nucleo
tides (1988) 8 (5-6): 1011-10.
14; (e) Brill et al., J. Am. Am. Ch
em. Soc. (1989) 111 (6): 2321-
2322.

In one example, the 3'end of the polynucleotide is modified to prevent reaction with a template-dependent DNA polymerase or to add a binding sequence. For example, by conjugating a dideoxynucleotide or ribonucleotide to oxidize ribose with periodate and then modifying the resulting dialdehyde by reductive amination with a large amine such as borohydride and aminodextran. R.

The predetermined amounts of reagents of the present invention can be provided in a packaged combination kit for convenience. A kit useful in the present invention for the determination of polynucleotides in a sample is a reagent for forming a target polynucleotide sequence from a polynucleotide, packaged with other reagents, 3 ′ thereof.
An extension probe having a sequence at the end which is hybridizable with the first sequence in the target polynucleotide sequence and a sequence homologous to the second sequence of the target polynucleotide sequence, wherein the second sequence is 5'of the first sequence. And non-adjacent), and a polydeoxynucleotide primer, which is labeled or has a group attached, allowing the sequence to be attached to a label or support. The kit further comprises a labeled polynucleotide probe capable of binding to the target polynucleotide sequence,
It contains any polynucleotide sequence required to modify the 3'end of the extender probe and not hybridized to the target polynucleotide sequence, and optionally a 3'-exonuclease. The kit is further packaged in combination with deoxynucleoside triphosphates such as deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxythymidine triphosphate. (DTTP) is contained. For use in multiple copy production methods, the kit may contain polydeoxynucleotide primers if the primers are not produced by degradation of the extension probe. The kit can also include polydeoxynucleotide polymerase and members of the signal producing system, and various buffering media, some of which include one or more of the reagents described above.

The relative amounts of the various reagents in the kit vary widely in order to provide a concentration of reagents that substantially optimize the reactions that occur in the method, and which also substantially optimize the sensitivity of the assay. Optionally, one or more reagents in the kit are provided as a dry powder (usually lyophilized and containing excipients) that dissolves to perform the assay of the invention. A reagent solution having a concentration suitable for is provided. Each reagent may be packaged in a separate container, or some reagents may be contained in one container as cross-reactivity and reagent life permit.

EXAMPLES The invention is further illustrated by the following specific examples. Temperatures are in degrees Celsius (° C) and parts and percentages are by weight unless otherwise indicated.

Example 1 Modification of extension probe by exonuclease digestion Oligodeoxyribonucleotide sequences 1 and 2: Polynucleotide extension probe; Oligomer 1 5'TGT TGT TCC GTT AGT TCG
TTT TAT TTG TCG AAA TCC
GCG ACC TGC TCC ATG TTA C
T3 '(SEQ ID NO: 1) and polydeoxynucleotide primer for amplification; Oligomer 2 5'TGT TGT TCC GTT AGT TCG
TTT TAT T3 ′ (SEQ ID NO: 2) is (“Oligonucleotide Synthesis: A Practical Approach”, edited by M. J. Gait, IRL Press (Ox
Ford, UK), 1984 T.S. Atkinson
And M.M. Synthetic by the phosphoramidite method (by Smith) and purified using denaturing polyacrylamide gel by standard method. Twenty-five bases at the 5'end of the extension probe, oligomer 1, was identical to the polydeoxynucleotide primer, oligomer 2, and was used to purify an amplifiable polynucleotide sequence having an intramolecular base pair structure.

Using the oligomer 1 to form the initial amplifiable structure and the oligomer 2 to carry out the amplification, the target polynucleotide bacteriophage M13m
p19 (duplex replication type, 7250 base pairs, Bethes
da Research Laboratories)
The DNA amplification protocol was used. 10 picomoles (pmol) of oligomer 1 and 600 molecules of M1
3mp19 is 10 mM KCl, 10 mM (NH4) 2
SO4, 20 mM Tris-HCl (pH at 25 ° C
8.8) 2 mM MgSO4, 0.1% Triton
X-100 and 20 nmoles of d
Mixed in NTP buffer. The reaction solution was heated at 95 ° C. to anneal the oligomer 1 of the extension probe to the template.
After denaturing for 5 minutes and cooling to room temperature, 5 units of T7
DNA polymerase (New England Bio)
labs), 5 units of T4 DNA polymerase (Bet
hesda Research Laboratori
es) or 4 units of Klenow fragment (US Bio
chemical) is added to the reaction mixture and the mixture is added at 37 ° C for 5
Was incubated for 10 minutes. During this incubation, the extended probe oligomer 1 that did not anneal to the target polynucleotide was cleaved by the 3'to 5'exonuclease activity of the enzyme. The extended probe annealed to the target polynucleotide was extended by the polymerase activity of the enzyme to form an amplifiable polynucleotide with an intramolecular base pair structure. T7,
T4 or Klenow polymerase then 95 ° C
The mixture was heat-inactivated by incubating at room temperature for 2 minutes, and the reaction solution was cooled to room temperature again. 100 pmol
e oligomer 2 and 1 to 2 units of Vent DN
A polymerase (NewEngland Biolab
s) was added again to a final volume of 100 microliters (μl). 90 ° C (30 seconds), 55 ° C (1
Min) and 72 ° C (5 min for the first 10 cycles and then 1.5 min) then a thermal cycler (Ericomp, Inc.) programmable for many cycles with the above three temperatures. ) Was used.
The liquid of these reactions is recovered at the end of the temperature cycle, 1x
1.2% agarose (Seakem GTG, FMC BioPr) in TAE buffer (40 mM Tris-acetic acid (pH 10.3 at 23 ° C), 10 mM EDTA).
analyzed by gel electrophoresis,
The NA product was visualized by staining the gel with ethidium bromide.

To confirm that the extension probe oligomer 1 which did not completely anneal to the target polynucleotide was removed from the reaction solution by treatment with 3'to 5'exonuclease, T4 polynucleotide kinase (U
A small amount of extended oligomer 1 labeled at 32P with SB) was included in the reaction. The liquid was collected after exonuclease incubation and analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. The results obtained from this experiment are summarized in Table 1.

[0118]

[Table 6]

The results in Table 1 show that DNA with 3'to 5'exonuclease activity was obtained after annealing of the extension probe oligomer 1 to the target polynucleotide, but before addition of the amplification polynucleotide primer and thermostable DNA polymerase. By treating the reaction solution with a polymerase, all the extension probe oligomer 1 was completely removed from the reaction solution, and sufficient poly- mer with an intramolecular base pair structure for amplification from 600 double-stranded DNA targets was obtained. This shows that it is possible to form nucleotides.

Example 2 Modification of extension probe by chain extension Oligodeoxyribonucleotide sequences 1 and 2: Polynucleotide extension probe; Oligomer 1 5'TAG CTA GCA GTA ACA TGG.
AGC AGT GTT GTT CCG TTA
GTT CGT TTT ATT TGT CGA A
AT CCG CGA CCT GCT CCA TG
T TAC T3 ′ (SEQ ID NO: 3) and polydeoxynucleotide plumer; Oligomer 2 5 ′ TGT TGT TCC GTT AGT TCG
TTT TAT T3 '(SEQ ID NO: 4) was synthesized by the phosphoramidite method and purified on a denaturing polyacrylamide gel. Included in oligomer 1 is the entire sequence of oligomer 2 (24 to 48 bases) used to generate amplifiable polynucleotides with an intramolecular base pair structure. Oligomer 1 from 9 to 23
And 65 to 79 bases are a 15 base pair stem and 41 bases.
It contains an inverted repeat sequence that can form an intramolecular base-pair structure consisting of a loop of bases. The 5'end 8 bases of oligomer 1 are the target polynucleotide, namely bacteriophage M.
It is not complementary to 13mp19.

Bacteriophage M13mp19 (double-stranded replicative, 7250 bases) was prepared using the extended probe oligomer 1 to form the first amplifiable structure with an intramolecular base pair structure and the oligomer 2 to carry out amplification. (Pair) DNA amplification protocol was used. 10 picomoles (pmol) of oligomer 1, 200 picomoles of oligomer 2 and 600 molecules of M13mp19
mM KCl, 10 mM (NH4) 2 SO4, 20 m
M Tris-HCl (pH 8.8 at 25 ° C), 2 m
Mixed in a buffer of M MgSO4, 0.1% Triton X-100 and 20 nmoles of dNTPs. Targeting the extension probe oligomer 1 DN
To anneal to A, the reaction was denatured at 95 ° C for 5 minutes and cooled to room temperature. 1 to 2 units of Ven
t DNA polymerase (NewEngland Bi
olabs) was then added to a final volume of 100 microliters (μl). The reaction solution is 10 at 72 ° C.
Incubated for minutes. During this step, the extension probe oligomer 1 which annealed to the target polynucleotide was subjected to Vent to form an intramolecular base pair (pb) structure.
It was extended by polymerase and amplified by oligomer 2. The extended probe oligomer 1 that did not anneal to the target polynucleotide formed an intramolecular stem-loop containing a 15 bp stem and an 8 base 5'single-stranded overhang. This 5'overhang was filled in by Vent polymerase with complementary nucleotides. The modified extension probe oligomer 1 because the 8 bases filled in by Vent polymerase were not complementary to the target polynucleotide.
Becomes inactive as a primer. This extension probe, which anneals to the target, has an 8 base mismatch at the 3'end and is therefore not extended by Vent polymerase.

90 ° C. (30 seconds), 55 ° C. (1 minute) and 72 ° C. (5 minutes for the first 10 cycles, then 1.5 minutes) temperature cycling depends on the above three temperatures. It was performed using a programmable thermal cycler (Ericomp, Inc.) for many cycles. The liquid of these reactions is recovered at the end of the temperature cycle,
AE buffer (40 mM Tris acetic acid (pH at 23 ° C
10.3), 1.2% agarose (Seakem GTG, FMC BioProdu) in 10 mM EDTA).
cts) gel was analyzed by electrophoresis and the DNA product was visualized by staining the gel with ethidium bromide.

All unannealed extension probe Oligomer 1 was venated with deoxyribonucleotide triphosphates during the first incubation at 72 ° C.
To confirm that it was filled in by the action of t polymerase, a small amount of extension probe oligomer 1 labeled at the 5P end with 32P using T4 polynucleotide kinase (USB) was included in the reaction. After the first incubation at 72 ° C, the liquid was collected and analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. The results obtained from this experiment are summarized in Table 2.

[0124]

[Table 7]

The results in Table 2 show that 600 molecules of double-stranded DNA
Vent at the end of the first cycle of amplification in the presence of target
It is shown that the extended probe oligomer formed an internal base pair structure or a stem-loop with a 5'single-stranded overhang that was effectively filled by the polymerase, thereby rendering it unusable as a primer in the next round of amplification. The amplification primer efficiently amplified the base pair structure within the amplifiable molecule formed in the first cycle.

Example 3 Modification of Extension Probes with Oligonucleotides Containing Thiophosphate Detection of about 600 double stranded target molecules using single primer amplification was repeatedly shown using oligonucleotides containing degradable thiophosphates. Was done. Oligonucleotide (5
6 bases) acts as an extension probe when making an amplifiable stem-loop, and as a primer to perform amplification following nuclease treatment.

The synthesis of extension probe oligonucleotides was automated (4 column Bi
(Osearch 8750 DNA synthesizer). Manual oxidation followed by 0.1M tetraethylthiuram disulfide (TETD) (Appl in acetonitrile).
ied Biosystems, Inc. , Foste
r City, CA). Applie
The remaining base was added under normal coupling conditions according to the protocol of d Biosystems, February 1991, Booklet No. 58 to Users. In this example, two different extension probes with identical sequences, each differing only in the number and position of the internucleotide linkages of thiophosphate, were used in separate experiments.

The formation and amplification of the stem-loop molecule is carried out in an appropriate buffer (20 mM Tris-HCl, pH 8.8,
10 mM KCl, 10 mM (NH4) 2SO4, 2 m
MMgSO4 and 0.1% Triton X-10
0), dNTPs (200 to 300 micromolar), double-stranded target polynucleotide molecules (600 molecules of M13mp19) and extension probe (initial concentration 0.5 to 4 micromolar) in a 100 microliter reaction system. It was The reaction was heat denatured at 95 ° C for 5 minutes and annealed at 25 ° C (room temperature) for 15 to 20 minutes. DNA polymerase with strong 3'exonuclease activity was added (10 units of T7 DNA polymerase per 100 μl, New England B.
iolabs (NEB), Beverly Mas
s). The reaction was incubated at 37 ° C. so that the extension probes that annealed to the target had their strands extended, while all unannealed extension probes were cleaved to the position of the thio bond. The extension probe was radiolabeled at the 5'end to monitor degradation. Complete degradation of the unannealed extension probe up to the thio bond was obtained in about 1 minute, while the remaining sequences were more resistant to degradation by 15 minutes. After extension / degradation was complete, the reaction was again heat-treated at 95 ° C for 1-2 minutes, which inactivated the T7 polymerase and denatured the newly formed stem-loop molecule from the original target molecule. . A thermostable polymerase was added (Stratagene, San Diego.
o, 5 units per 100 μl of Pfu of CA) reaction is cycled as described in the previous example. These reaction solutions were 1 × TBE buffer (89 mM Tris-borate,
1.2 in 89 mM boric acid, 0.2 mM EDTA)
% Agarose (Seakem, EMCBioProdu
cts) gel analysis and amplification products were visualized by ethidium bromide staining.

Extension probe containing a thio bond: One thiophosphate bond between A34 and T35 (↓): a) 23 base primer remaining after degradation (underlined) 3'TC ATT GTA CCT CGT CCA
GCG CCTAAA GCT G T T T A
↓ T T T T G C T T G A T T G
CCTTGTTTGT 5 '(SEQ ID NO: 5) 1. Three thiophosphate bonds (↓) between T33 and T36: a) Three primers (24, 23, 2) remaining after degradation
2 bases) mixture (underlined)

The invention also relates in a second aspect to the general use of thiophosphate-containing oligonucleotides as primers for nucleic acid amplification.

Summary of Interpretation of the Invention As an embodiment of the present invention, a method of forming a polynucleotide sequence complementary to a single-stranded target polynucleotide sequence ("target sequence") is described. The method comprises: (a) hybridizing to a 3'end of a target sequence a polynucleotide primer having a 3'end containing a nucleotide monophosphate in which at least one phosphate oxygen has been replaced by sulfur.
(B) extending the polynucleotide primer along the target sequence, and (c) dissociating the extended polynucleotide primer from the target sequence.

Another embodiment of the invention is a polynucleotide having a sequence identical to the single stranded target polynucleotide sequence ("target sequence"), except that a sulfur bond to phosphorus is present instead of oxygen. It is a method of forming an array. The method comprises: (a) hybridizing a first polynucleotide primer ("first primer") containing at least one phosphorus bond to the 3'end of the target sequence, and (b) the first primer along the target sequence. A second polynucleotide primer that is the same or different from the first primer (“the second primer”) that is extended and (c) dissociates the extended first primer from the target sequence, and (d) to the 3 ′ end of the extended first primer. )
And (e) when the first and second primers are the same, the extended first primer is the template for the first primer, and when the first and second primers are different, the extended first primer Is a template for the second primer and the extended second primer comprises extending the second primer along the extended first primer to form an extended second primer such that the extended second primer is the template for the first primer. .

The methods of the invention apply to methods of making multiple copies of a target polynucleotide sequence. In general,
These methods include forming along the target sequence or an extended polynucleotide primer an extension product of the polynucleotide primer such that the extension product is a copy of the target sequence. The improvements provided by the present invention include primers with a 3'end consisting of a nucleotide monophosphate containing at least one phosphorus-sulfur bond.

Accordingly, an embodiment of this aspect of the invention is a method of producing multiple copies of a polynucleotide sequence. The method comprises: (a) (1) a single-stranded polynucleotide having such a polynucleotide sequence and flanked by at least partially complementary first and second flanking sequences at each end, (2) a single strand A polynucleotide primer comprising at least one thiophosphate bond at the 3'end of a chain polynucleotide, the polynucleotide primer having a portion of at least 10 bases hybridizable at its 3'end to the bases of the first and second flanking sequences. ,
(3) nucleotide triphosphate, (4) preparing in combination with a template-dependent polynucleotide polymerase, and (b) wholly or partially continuously or coexistently (1) complementary Dissociating the single-stranded polynucleotide from the sequence, (2) hybridizing the polynucleotide primer with a sequence adjacent to the 3'end of the single-stranded polynucleotide, (3) providing a first extended polynucleotide primer To extend the polynucleotide primer along the single-stranded polynucleotide, (4) dissociate the first extended primer and the single-stranded polynucleotide, (5) the first extended polynucleotide primer and the polynucleotide Hybridizing the primer, (6) providing a second extension polynucleotide primer Extending the polynucleotide primer along the first extension primer for
(7) Dissociating the second extension polynucleotide primer from the first extension polynucleotide primer, and (8) incubating this combination under the condition that steps (5) to (7) above are repeated. .

In accordance with this aspect of the invention, there is provided a method of forming multiple copies of a single stranded target polynucleotide sequence containing at least one phosphorus-sulfur bond ("target sequence"). The method comprises: (a) hybridizing a first polynucleotide primer containing at least one phosphorus-sulfur bond to the 3'end of the target sequence ("first primer"), and (b) first along the target sequence. Extending a primer (wherein the first primer can hybridize to (1) an extended first primer, or (2) an extended second polynucleotide primer ("second primer"), and extend along it; The extended second primer hybridizes to a sequence complementary to the target sequence (complementary sequence) and can be extended by extension of the second primer), (c) dissociates the extended first primer from the target sequence (D) hybridizing the first or second primer to the 3'end of the extended first primer, (e) the extended first primer Extending the first or second primer along the lymer, (f) dissociating the extended first primer or the extended second primer from the extended first primer, (g) the extended first or second It consists of hybridizing the first primer to the 3'end of the primer and repeating steps (h) (e) to (g).

Another embodiment according to this aspect of the invention is a method of forming multiple copies of at least one double-stranded polynucleotide sequence (“polynucleotide sequence”). The sequence comprises a single-stranded target polynucleotide sequence (“target sequence”) and its complement. The method comprises: (a) a sample expected to contain one or more double-stranded polynucleotide sequences in a sample, provided that the primer hybridizes to the target and complementary sequences and extends along the primer. Is treated with a polynucleotide primer capable of hybridizing to each target and complementary sequence expected to be present in, where the extension product formed from one primer is dissociated from its complementary strand to the other. A primer is selected that can act as a template for the formation of extension products of the primer, wherein at least one primer for each of the double-stranded polynucleotide sequences comprises at least one phosphorus-sulfur bond, (b) single-stranded If the sequence is present to create the molecule, then primer extension products are generated from the template. And (c) under conditions such that a primer extension product is formed using the single strand created in step (b) as a template, resulting in amplification of the target sequence and, if present, the complementary sequence. Then, the single-stranded molecule prepared in the step (b) is treated with the primer in the step (a).

Another interpretation of the invention is a method of detecting the presence or absence of at least one specific polynucleotide sequence (“polynucleotide sequence”) in a sample that is expected to contain the polynucleotide sequence. The method comprises (a) one or more polynucleotide sequences for each different polynucleotide strand to which the primer is hybridized, provided that an extension product of each primer is formed that is complementary to that strand. Treatment of the sample expected to contain with one polynucleotide primer (“primer”) for each polynucleotide sequence expected to be present in the sample, wherein the extension product formed from one primer is A primer is selected that, when dissociated from its complementary strand, can serve as a template for the formation of extension products of the other primer, and at least one primer for each double-stranded polynucleotide sequence is at least one phosphorous. -If a (b) sequence containing a sulfur bond is present, Dissociating the primer extension product from its template to create a double-stranded molecule, (c) the primer extension product is formed using each single strand made in step (b) as a template, and the target sequence and Treating the single-stranded molecule made in step (b) with the primer of step (a) under conditions that result in the amplification of complementary sequences, if any, (d) the product of step (c) To adding a labeled oligonucleotide probe capable of hybridizing to the sequence for each sequence detected or a variant thereof, and (e) determining if such hybridization occurs.

Another interpretation of the invention is a method of determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte. The method comprises: (a) forming a single-stranded polynucleotide flanking at least 80% complementary first and second flanking sequences, each consisting of at least 15 nucleotides, as a result of the presence of the analyte;
(B) forming an extension product of a polynucleotide primer at least at the 3'end of a polynucleotide primer capable of hybridizing to a flanking sequence at the 3'end of a single-stranded polynucleotide sequence in the presence of nucleotide triphosphate and a template-dependent polymerase Wherein the steps (a) and (b) are performed wholly or partly in succession or concomitantly, and (c) a sequence identical and / or complementary to the polynucleotide sequence. Detecting an extended polynucleotide primer comprising According to the above method, in the step (a), S2 is 5'of S1.
Variants may be included in which the extension probe is combined with an analyte having non-contiguous non-complementary nucleotide sequences S1 and S2 that are flanking and that are at least 10 nucleotides in length. The extension probe has a sequence S3 capable of hybridizing to S1 and a sequence S4 homologous to S2 on the 5'side of S3 and further capable of extending along said analyte.

The invention also includes (a) at least one phosphorus-
It includes a kit comprising a polynucleotide primer having a 3 ′ end consisting of a nucleotide monophosphate containing a sulfur bond, (b) a nucleoside triphosphate and (c) a template-dependent polynucleotide polymerase all together. The kit for amplification of the target polynucleotide sequence can also include a second polynucleotide primer. Involved is a primer in which one extension product along the target sequence acts as a template for the other extension. In the above kit, the second primer has a 3 ′ end containing a nucleotide monophosphate containing at least one phosphorus-sulfur bond.

Brief Description of the Figures Figures 9 to 11 are overviews of different embodiments according to the invention.

FIGS. 12-14 are photographs of agarose gels of the reaction products obtained in the Examples section below.

In the accompanying drawings, these are referred to as a second interpretation of the invention.

DESCRIPTION OF SPECIFIC EMBODIMENTS In its broadest sense, the present invention provides for the formation of polynucleotide sequences complementary to single-stranded target polynucleotide sequences. In the method, a polynucleotide primer having a 3'end containing a nucleotide monophosphate in which at least one phosphate oxygen has been replaced by sulfur is hybridized to the 3'end of the target polynucleotide sequence. The polynucleotide primer is extended along the target polynucleotide sequence and the extended polynucleotide primer is dissociated from the target polynucleotide sequence.

Up until now, oligonucleotides containing thiophosphate groups were considered to be poor substrates for polymerases. See, for example, Gao et al., Supra.
As a result of the present invention, synthetic oligonucleotide primers resistant to degradation by the exonuclease activity of many DNA polymerases are used in the amplification process. Therefore, polymerases with 3'to 5'exonuclease activity are used in various extension and amplification processes. The editing function provided by this exonuclease activity confers higher fidelity of replication in the amplification process.

The method involves the amplification of a polynucleotide sequence such as by the polymerase chain reaction cited above, single primer amplification and amplification using a random sequence oligonucleotide as a primer, in which the polynucleotide sequence derived from the sample is amplified. It has particular application in the field of amplification. This method provides a very convenient method for amplifying nucleic acid sequences.

As a result of the present invention, synthetic oligonucleotide primers are used in extension procedures involving amplification of nucleic acids regardless of degradation by the exonuclease activity associated with many DNA polymerases used in nucleic acid extension and amplification procedures. For example, PCR leading to exponential amplification of nucleic acids is performed using an enzyme other than Taq polymerase, which does not have 3'-5 'exonuclease activity, and therefore cannot remove the mismatch. Many amplifications by this method have been described, some requiring temperature cycling and others amplifying at a single temperature.
The present invention provides an amplification process such as PCR performed using a single enzyme. Another advantage of the present invention is the scope of detection of genetic polymorphisms or point mutations in diagnosing routinely described genetic diseases using PCR. In order to introduce an artificial restriction enzyme site by a primer having a mismatch at the 3'end or to detect a single base mismatch in the target DNA, a primer having an enzyme mismatch regardless of-
Used with enzymes that have 3'exonuclease activity to digest the template region.

Before proceeding further with the description of particular embodiments of the invention, a number of terms are defined. (All other terms are as defined above.)

Target Polynucleotide Sequence--A sequence of nucleotides to be identified, usually found in a polynucleotide analyte, the identity of which is the variety of primers and other primers necessary to effect amplification of the target sequence contained therein. It is known to be somewhat sufficient for the preparation of the molecule. Generally, the primer hybridizes to and extends along the target polynucleotide sequence, thus the target polynucleotide sequence acts as a template. The target polynucleotide sequence is usually not required, but will lie between two defined sequences. Generally, primers and other probe polydeoxynucleotides are defined sequences, or at least a portion of such target sequence, at least 10 at their 3'ends.
A fragment of nucleotides, preferably 15, often 2
It hybridizes to a fragment of 0 to 50 nucleotides.
As mentioned above, the target polynucleotide sequence is generally two fixed, non-contiguous, complementary or non-complementary nucleotide sequences, S1 and S2, which are capable of hybridizing to such a primer or probe. Hold. The target polynucleotide sequence is usually about 30 to 50
It contains 00 or more nucleotides, preferably 50 to 1000 nucleotides. The two non-contiguous, non-complementary nucleotide sequences S1 and S2 preferably each contain 10 to 100 nucleotides and are separated by at least 10 bases, preferably at least 100, usually 200 to 10000 bases. The target polynucleotide sequence is part of many polynucleotide analytes. The target polynucleotide sequence is generally a fragment of a larger molecule, or essentially the entire molecule. The minimum number of nucleotides in the target polynucleotide sequence is selected to ensure that the presence of the target polynucleotide sequence in the sample is a specific indicator of the presence of the sample polynucleotide analyte. Very roughly, the length of a sequence is usually about 1.6 log L nucleotides, where L is the number of base pairs in the genome of the biological sample. The maximum number of nucleotides in the target sequence is generally due to polynucleotide analytes and truncation, or during separation and other procedures necessary to prepare samples for efficiency of assay and sequence detection and / or amplification. It is governed by the length of the decomposed part.

Polydeoxynucleotide primer--
A polydeoxynucleotide, which is a single-stranded normally synthesized deoxynucleotide containing a sequence capable of hybridizing at its 3'end to a sequence having a determined target polynucleotide sequence. Usually, polydeoxynucleotide primers have at least 50%, preferably 70%, more preferably 90%, and most preferably 100% complementarity as a determined sequence. The number of deoxynucleotides in the hybridizable sequence of the polydeoxynucleotide plumer is determined so that the stringent conditions used to hybridize the polydeoxynucleotide primers prevent extremely random non-specific hybridization. Usually, the number of deoxynucleotides in the polydeoxynucleotide primer is at least the same as the determined sequence of the target polynucleotide sequence, ie at least 10 deoxynucleotides, preferably at least 15 deoxynucleotides and generally about 10 to 200, preferably 20 to 50 deoxynucleotides.

Methods for Extending a Primer--Polynucleotide Polymerase or at least 3 ′ of the primer
A single-stranded template polynucleotide having a sequence distinct from its 3'end that is hybridizable to either or both ends. Methods for extending the primer also include nucleotide triphosphates or analogs thereof that can act as substrates for enzymes and other substances and divalent metal ions (usually magnesium), pH, ionic strength, organic solvents (such as formamide), etc. Conditions necessary for enzyme activity such as

According to the present invention, at least one polydeoxynucleotide primer used for extension along a template or target polynucleotide comprises a nucleotide monophosphate in which at least one phosphate oxygen is replaced by sulfur. It has an edge. Preferably 1 to 5 phosphoric acid oxygens are replaced by sulfur, more preferably 1 to 2 phosphoric acid oxygens. Sulfur is mostly attached to phosphorus alone (thiophosphate group), but it can also be attached to the carbon atom of ribose or the carbon atom of the label. Therefore, at least one polydeoxynucleotide primer that can be used in the method of the present invention, preferably 1 to 5
, More preferably 1 to 2 phosphorus-sulfur bonds. A consideration for the number of phosphorus-sulfur bonds is the degree of complementarity between the primer and template polynucleotide. In general, the greater the complementarity, the greater the allowable number of phosphorus-sulfur bonds. Phosphorus-sulfur bonds are commonly found at the 3'end of polydeoxynucleotide primers,
Usually present in the last 10 nucleotides of the 3'end, preferably in the last 5 nucleotides of the 3'end, more preferably in the last nucleotide of the primer. These sulfur-containing polydeoxynucleotide primers are prepared according to known techniques, as described below.

In an embodiment of the invention, methods for forming a polynucleotide sequence complementary to a single stranded target polynucleotide sequence (“target sequence”) are described. The method comprises: (a) hybridizing to a 3'end of a target sequence a polynucleotide primer having a 3'end consisting of a nucleotide monophosphate in which at least one phosphate oxygen has been replaced by sulfur. Extending a polynucleotide primer along a target sequence, and (c)
It consists of dissociating the extended polynucleotide primer from the target sequence.

A specific example of such a method is schematically shown in FIG. Primer P1 has a phosphorus-sulfur bond present at the 3'end (indicated as -S in the figure). The primer hybridizes with T1 of the target polynucleotide sequence (T). The primer is extended along T to produce extension primer H, which is complementary to T. For example, T
A T for which a particular sequence such as 2 is known is selected.
The extension primer contains the sequence P2 complementary to T2. In addition, the extension primer is dissociated from the complex.

Another embodiment of the invention forms a polynucleotide sequence having a sequence identical to the single stranded target polynucleotide sequence except that a phosphorus-sulfur bond is present instead of a phosphorus-oxygen bond. Is the way to do it. The method is
(A) hybridizing a first polynucleotide primer containing at least one phosphorus-sulfur bond ("first primer") to the 3'end of the target polynucleotide sequence, and (b) along the target polynucleotide sequence. Extending the first primer, (c) dissociating the extended first primer from the target polynucleotide,
(D) hybridizing to the 3'end of the extended first primer with a second polynucleotide primer ("second primer"), which is the same as or different from the first primer, and (e) the first and second primers Are the same, the extended first primer is the template of the first primer, when the first and second primer are different, the extended first primer is the template of the second primer,
The step of extending a second primer along an extended first primer to form an extended second primer such that the extended second primer serves as a template for the first primer. The method comprises dissociating the extended second primer from the extended first primer. Conditions are chosen in order to obtain a repetitive cycle in which the extended primer is dissociated from its template and the primer hybridizes to and extends along the extension primer. The circulation is repeated at least three times. Extension is carried out in the presence of nucleotide triphosphate and template-dependent polynucleotide polymerase.

The methods of the invention are useful, for example, in polynucleotide amplification, such as PCR and single primer amplification in which one or more copies of the target polynucleotide sequence, ie, a sequence identical to the target polynucleotide sequence, is formed. It is clear that there is.

The use of this method for single primer amplification is shown in FIG. In the description below, the appropriate conditions for the steps of hybridisation, extension and dissociation are chosen as discussed below. The polydeoxynucleotide primer P1 hybridizes to T1, which is capable of hybridizing to T2 of the target polynucleotide sequence and is preferably complementary to T2. P1 can also include a label W (not shown). P1 is the sequence P1 and T2
And an extension primer H containing P2 which is complementary to P1
Hybridizes to the target T to form and is extended along. H is dissociated from T and P1, hybridized to P2 of H and the T1 portion of T, and extended along H and T, respectively. H'and H respectively
To obtain, the duplex is dissociated and P1 hybridizes to H and T and is extended along them. Further iterations will result in multiple copies of the target polynucleotide sequence being detected due to the presence of label W.

Another embodiment of this interpretation of the invention is shown in FIG.
1, each primer having a nucleotide monophosphate containing at least one phosphorus-sulfur bond is PC
Used for R amplification. Of course, one has a nucleotide monophosphate containing at least one phosphorus-sulfur bond, but it is within the scope of the invention to perform PCR with two primers. As mentioned above, the PCR method is used to form multiple copies of at least one double-stranded polynucleotide sequence. The sequence comprises a single stranded target polynucleotide sequence and its complement. Samples expected to contain one or more double-stranded polynucleotide sequences are polynucleotide primers P'1 and P'2 capable of hybridizing to the target expected to be present in the sample and its complementary strand, respectively. Is processed in. Conditions are selected in which the primer hybridizes to the target and complementary strand along which the primer extends. When dissociated from its complementary strand, the extension product formed from one primer can act as a template for the formation of the extension product of the other primer. As seen in FIG. 11, the complementary sequences are T ′ and T ″. T '
Has the sequences T'1 and T'2 and T "is the sequence T"
1 and T ″ 2. P'1 hybridizes with T'1 of T'and P'2 hybridizes with T "2 of T". Under appropriate conditions, P'1 is extended along T'to obtain V and P'2 is extended along T "to obtain V '. P'1 and T'1 are complementary, P'2 and T "2 are complementary, P'1 and T" 1 are homologous, and P'2 and T'2 are homologous. Is clear from FIG. The extension primer is dissociated from each of these templates. V, respectively
The primer P'1 molecule hybridizes to and is extended along T'and V'respectively to obtain W ', W and V', and the primer P'2 molecule hybridizes to T "and V respectively. , Stretched along it. As seen in FIG. 11, repeating the cycle results in multiple copies of W and W ′ containing the target sequence. Cycles are conducted under conditions such that the primer extension product is formed using the single strand produced as template and, if present, amplifies the target sequence and its complementary sequence.

The method is applied when the target polynucleotide sequence is DNA or RNA. In one interpretation, the polydeoxynucleotide primer is labeled with a label (reporter molecule). Reporter molecules are eg
A detectable group or binding agent such as biotin or a nucleotide sequence separate from the sequence that hybridizes to the target sequence. The reporter molecule attaches directly to the thiophosphate group of the primer, or elsewhere. The extension primer is detected by the reporter molecule covalently attached to the probe. A probe usually has a nucleotide sequence that is homologous or complementary to a portion of the target nucleotide sequence that differs from the sequence to which the primer binds.

Another embodiment of the invention relates to a method of detecting the presence of a polynucleotide analyte in a sample suspected of containing the polynucleotide analyte. The medium containing the sample, if present, forms a single-stranded target polynucleotide sequence from the polynucleotide analyte,
It is processed as described above. The target polynucleotide sequence has two non-contiguous, non-complementary sequences S1 and S2, where S2 is 5'of S1 and is at least 10 nucleotides in length. The medium is combined with an extension probe having two deoxynucleotide sequences. Such an approach for single primer amplification is described above. The sequence at the 3'end of the extension probe (EP1) is hybridized to S1. Deoxynucleotide sequence (EP
The other of 2) is homologous to S2. A polydeoxynucleotide sequence capable of hybridizing to a nucleotide sequence complementary to S2 is included where modification of the extension probe does not provide a primer. The extension probe is present at a concentration below the concentration of the primer, usually below 1%. According to the present invention, the primer has a 3'end consisting of a nucleotide monophosphate in which at least one phosphate oxygen is replaced by sulfur. Deoxynucleotide triphosphates and one or more polydeoxynucleotide polymerases are also combined. (1) an extension probe hybridizes to and extends along the target polynucleotide sequence to form a duplex, (2) an extension probe that does not hybridize to the target polynucleotide sequence is modified, (3) The extension probe is dissociated from the duplex,
(4) the primer is hybridized to and extended along the extended sequence to form a second duplex containing the extended primer, (5) the extended primer is dissociated from the duplex, and (6) Conditions are selected such that the primer hybridizes to and extends along the extension primer to form a duplex containing the extension primer. Steps (5) and (6) are repeated, steps (a) and (b) being performed concomitantly or wholly or partially in succession. A test is then made for the presence of the extension primer, the presence of which is indicative of the presence of the polynucleotide analyte. Steps (5) and (6) are performed at least three times, preferably at least 10
Repeated repeatedly; usually, the number of repetitions is preferably 30 times or less. Generally, steps (5) and (6) are repeated a sufficient number of times to provide accurate detection of the polynucleotide analyte. When the polynucleotide analyte is RNA, the polydeoxynucleotide polymerase comprises reverse transcriptase.

Conditions, concentrations, etc. for carrying out these methods are similar to those described above unless otherwise stated.

Single Primer Amplification or PCR
, The method is carried out for a time sufficient to obtain the desired copy number of the extension primer or its complement. This depends, for example, on the purpose for which the amplification is to be carried out, eg in the assay of polynucleotide analytes. In general,
The method is performed for about 1 to 10 minutes per cycle, and the number of cycles is about 1 to 200 times or more,
Usually 5 to 80 times, often 10 to 60 times.
For convenience, it is usually desirable to minimize the time and number of cycles. In general, the time for a given degree of amplification is shortened, for example, by choosing a concentration of nucleotide triphosphate sufficient to saturate the polynucleotide polymerase and increasing the concentration of the polynucleotide polymerase and polynucleotide primer. To be done. Generally, the time for carrying out the method is about 5 to 200 minutes. For convenience, it is usually desirable to minimize time.

As stated above, the main advantage of the present invention is 3 '.
A template-dependent polynucleotide polymerase with exonuclease activity is used for amplification of the polynucleotide. The concentration of this polymerase is chosen to be sufficient to complete chain extension. The concentration of template-dependent polynucleotide polymerase is usually determined empirically. Preferably, a sufficient concentration is used so that the increase in concentration is not less than 5-fold, and preferably the time for 2-fold amplification is not reduced. The main limiting factor is generally the price of the reagent.

Copies of extension primers are detected in many ways. For example, in the method, the polydeoxynucleotide primer molecule is labeled with a reporter molecule such as a ligand, a small organic molecule, a polynucleotide sequence, a protein, a support, an operator-promoter pair, an intercalating dye and the like. Standard methods of specifically detecting nucleic acid sequences are used. According to the present invention, sulfur atoms are incorporated into the amplification product, so that a reporter molecule can be constituted, which is an entity related to the presence of the target polynucleotide. In this approach, the amplified product is chemically treated in many ways to detect the presence of sulfur atoms. For example, the reactants according to the invention are treated with an enzyme that cleaves the double stranded product on the opposite side of the thiophosphate bond. Such enzymes are, for example, Hinc I
I. In another approach, the reaction product is chemically treated by known methods to cleave the thiophosphate bond. Other methods of detecting the presence of sulfur atoms in reaction products have been proposed in the art.

One method of detecting nucleic acids is to use nucleic acid probes. The method using the probe is described in US Patent Application No. 773,386, US Pat. No. 4,868,810 of September 6, 1985, which is incorporated herein by reference.
4 are described.

Other assay and detection methods are described in US patent application Ser. Nos. 07 / 299,292 and 07/3 of January 19, 1989 and August 29, 1989, respectively.
99,795. Also, EP37
See 9369 and U.S. patent application Ser. No. 07 / 555,323 of July 19, 1990 and EP 469755. These applications are incorporated herein by reference.

Examples of specific labels or reporter molecules and their detection have been described above. Signal detection is described above. Various techniques are used to prepare polydeoxynucleotide primers or other polynucleotide sequences, as described above.

Polydeoxynucleotide primers containing at least one monophosphate with a 3 ′ end consisting of a nucleotide monophosphate in which the oxygen of at least one phosphate is replaced by sulfur are prepared according to known techniques. . For extension probes containing such groups, see specific examples as described above.

For convenience, the predetermined amount of reagents used in the present invention are provided in a packaged combination kit. The kit comprises (a) a polynucleotide primer having a 3 ′ end consisting of a nucleotide monophosphate containing at least one phosphorus-sulfur bond, (b) a nucleotide triphosphate and (c) having exonuclease activity, or having No template-dependent polynucleotide polymerase is included as a packaging combination. A kit for the amplification of a target polynucleotide sequence comprises the above items, and in order to carry out PCR, the primer extension product of the primer along said target sequence acts as a template for the extension of the other primer It includes a second polynucleotide primer as related. The second primer also has a 3'end consisting of a nucleotide monophosphate containing at least one phosphorus-sulfur bond.

For assaying a polynucleotide analyte in a sample, a kit useful in the present method includes, in a package with the other reagents described above, a reagent for forming a target polynucleotide sequence from the polynucleotide analyte. . further,
Is the polydeoxynucleotide primer labeled,
Alternatively, groups are provided to provide labeled or support-bound sequences. The kit further comprises a labeled polynucleotide probe capable of binding to the amplified target polynucleotide sequence. The kit is further packaged in a package such as deoxyadenosine triphosphate (dATP),
Includes deoxynucleotide triphosphates such as deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP) and deoxythymidine triphosphate (dTTP) deoxynucleotide triphosphates. The kit further comprises a signal production system and various buffers containing one or more of the above reagents.

The relative amounts of the various reagents in the kit are such that they provide the reagent concentrations that essentially optimize the reaction needed to bring about the method and essentially optimize the sensitivity of the assay. Can be widely changed. A dry powder containing excipients, provided by dissolving, in a suitable environment, one or more reagents of the kit in a reagent solution having a suitable concentration for performing the method or assay according to the present invention, Usually, it is provided as a lyophilized product. Each reagent is packaged in a separate container, or several reagents that allow cross-reactivity and shelf life are combined in one container.

Examples The second interpretation of the present invention is further illustrated by the following specific examples. Unless otherwise noted, temperatures are in degrees Celsius (° C) and parts and percentages are by weight.

Example 1 Preparation of a 415mer with inverted repeats A preparation of a 415mer with inverted repeats (stem-loop molecules) was prepared in US patent application Ser. No. 07 / 555,323 filed Jul. 19, 1990. It was done according to the method described. See also related publication EP 469755, which is incorporated herein by reference. For preparation, 10 mM Tris-Cl, pH 8.3, 50 m
10 atmole of single chain M13mp19 in 100 microliter reaction system containing M KCl, 1.5 mM MgCl 2, 0.01% gelatin and 200 micromolar deoxynucleotide triphosphates (dNTPs).
(Target DNA, Bethesda Research
Laboratories (BRL) were used. 1
Micromolar single primer amplification primer ON
1 (polydeoxynucleotide primer) and 0.
In the presence of 1 micromolar extension probe ON2, the reaction solution was heat-treated at 95 ° C. for 5 minutes and cooled to room temperature for 15 minutes. This causes the extension probe to anneal to the target DNA. Taq polymerase (Stratag
ene, Inc. , San Diego, CA) (5
Unit) was added and the temperature was cycled as follows: 90 °
C30 seconds, 55 ° C 60 seconds, 72 ° C 90 seconds. This temperature cycle was repeated 30 times. The 415-mer product was visualized by agarose gel electrophoresis. Agarose gel is SeaKem LE agarose (FMC BioP
products, Rocklands, ME) at 1.2% weight / volume (w / v). The reaction mixture (10 microliters) was 6xdye (0.25% promophenol blue, 0.25% xylene cyanol and 15% ficoll, type 400 (Maniat).
"Molecular Cloning, Laboratory Manual" by is et al. 45
5 (1982))) and subjected to electrophoresis in 1 × TBE buffer (0.1 M Tris, 89 mM boric acid, 2 mM sodium ethylenediaminetetraacetate). The band constituting the 415mer product was identified by ethidium bromide (1 mg / ml) staining and detected by testing under UV light. The gel is Fotodyne
, Inc. (New Berlin, WI) Polaroid MP- equipped with UV transilluminator
Photographed using a 4-land camera. 415mer
Reactions that gave a single band corresponding to were collected by standard methods ("Current Protocols in Molecular Biology", Section 15).
Ethanol precipitation according to Chapter, page 15.2.2) and quantification using a photometer.

Oligonucleotides ON1 and ON2 were as follows: ON1: 5′TGT TGT TCC GTT AGT.
TCG TTT TAT T3 '(SEQ ID NO: 2) ON2: 5'TGT TGT TCC GTT AGT
TCG TTT TAT TCA TAG TTA
GCG TAA CGA TCT AAA GTT T
TG TC3 '(SEQ ID NO: 7)

Example 2 Amplification with a primer having a thiophosphate in the second position from the end of the 3 ′ The 415mer produced as described in Example 1 was more fully described as follows: It was used as the target sequence in a single primer amplification reaction with several different primers containing one or more phosphorus-sulfur bonds instead of phosphorus-oxygen bonds. Primer 1 was ON1 with a phosphorus-sulfur bond between the end of the 3'end and the penultimate end as shown in bold below: 5 '
TGTTGT TCC GTT AGT TCG TT
T TAT T3 '(SEQ ID NO: 8). Primer 1 was prepared for Primer 2 as described below.

Automatically up to the position of the thio-modified bond shown in bold type (Millipore Corp., Bedfo
rd, MA 4 column Biosearch 8750D
NA synthesizer) Synthesized next oligonucleotide (ON
3): 5'TGT TGTTCC GTT AGT T
CG TTT TAT TCA TAG TTAGCG
TAA CGA TCT AAA GTT TTG
Primer 2 was prepared by exonuclease digestion of TC3 '(SEQ ID NO: 9). Manual oxidation was followed by 0.1M tetraethylthiuram disulfide (TETD) in acetonitrile (Applied Biosystem).
s, Inc. , Foster City, CA). The remaining bases are February 1991 Appli
edBiosystems, Inc. User Bulletin No. 58 was added under normal coupling conditions. The above oligonucleotide (2
μM) digestion was performed in 1 × Vent buffer. The reaction was heat denatured at 95 ° C for 5 minutes and then cooled to room temperature. T7 DNA polymerase, unmodified type II (New
England Biolabs, Beverly,
MA) was added and the reaction was incubated for 5 minutes at 37 ° C. T7 was then inactivated by heat treating for 2 minutes at 95 ° C. The resulting reaction solution containing Primer 2 was used in the following experiments. Primer 2:
5'TGT TGT TCC GTT AGT TCG
TTT TAT T3 '(SEQ ID NO: 10).

The amplification reaction was performed according to the following protocol: 415mer was about 500 molecules of 415me.
It was serially diluted with water to obtain a medium containing r. V
ent polymerase buffer (1x) (100 mM KC
1, 200 mM Tris-HCl, pH at 25 ° C
8.8, 100 mM (NH4) 2SO4, 20 mM M
gSO4, 1% Triton X-100), 300
μM dNTPs and 1 μM Primer 1 were combined. The mixture was heat treated at 95 ° C for 5 minutes and then cooled at room temperature for 15 minutes. Taq DNA Polymerase (Stratagene, San Diego, CA)
(5 units) was added to the reaction system and subjected to 50 cycles of temperature cycling as follows: 72 ° C. 5 min (1 time), 90
30 seconds at 55 ° C, 60 seconds at 55 ° C and 90 seconds at 72 ° C (50
Times). As a control, ON1 which does not have a phosphorus-sulfur bond instead of a phosphorus-oxygen bond, that is, 5'TGT
TGT TCC GTT AGT TCG TTT T
The above amplification was repeated using the control primer for AT T3 '(SEQ ID NO: 2). The results of the above experiments were determined by agarose gel electrophoresis as described above and are shown in Figure 12. Here, the control reaction is shown in lane 1 and the reaction with primer 1 is shown in lanes 2 and 3.

Amplification of the 415mer was also performed using primer 2 and a protocol similar to the method described above. The concentration of primer 2 is 1 μM and 2.5 units of Pf
uDNA polymerase (Stratagene, San
Diego, CA) was used instead of Taq DNA polymerase. Results are shown in FIG. Lane 1 represents a negative control with no target molecule present. Lanes 2, 3 and 4 are according to the invention 41
Representing amplification of about 300, 30 and 3 molecules of 5 mer, respectively, this serial dilution was used to obtain a concentration of 415 mer before amplification.

Example 3 Single Primer Amplification with an Extended Probe Containing a Degradable Thiophosphate to Generate a Primer In Situ Detection of approximately 600 molecules of double stranded target using single primer amplification is degradable Repeatedly shown with an oligonucleotide containing thiophosphate. Oligonucleotide (56
The base) acts as an extension probe that produces an amplifiable stem-loop and, after nuclease treatment, acts as a primer for amplification.

The synthesis of the extension probe oligonucleotide was carried out automatically up to the position of the thio-modified bond (4 column B
iosearch 8750 DNA synthesizer). Manual oxidation followed by 0.1M tetraethylthiuram disulfide in acetonitrile (TETD) (Applied)
Biosystems, Inc. , Foster Ci
ty, CA). The remaining base is Appl
ied Biosystems was added under conventional coupling conditions according to the protocol of User Burtin No. 58, February 1991.

Extension probe containing a thio bond: Three thiophosphate bonds between T33 and T36 (↓): Three types of primers remaining after degradation (24, 23, 22)
Base) (underlined) mixture

The formation and amplification of the stem-loop molecule is carried out in an appropriate buffer (20 mM Tris-HCl, pH 8.8, 1,
0 mM KCl, 10 mM (NH4) 2SO4, 2 mM
MgSO4 and 0.1% Triton X-10
0), dNTPs (200 to 300 micromolar), double-stranded target polynucleotide molecule (M13mp1
9 in 600 molecules) and an extension probe (initial concentrations 0.5 and 1 micromolar) in a 100 microliter reaction system. The reaction was heat denatured at 95 ° C for 5 minutes and annealed at 25 ° C (room temperature) for 15 to 20 minutes. Each of the three DNA polymerases with strong 3 ′ exonuclease activity was added separately (6 units of Klenow DNA polymerase per 100 μl, 5 units of T4 DNA polymerase per 100 μl (New England Biolabs (N
EB), Beverly Mass) or 100 μl
5 units of T7 DNA Polymerase (New En
ground Biolabs (NEB), Beverl
y Mass)). The reaction was incubated at 37 ° C. so that the extension probes that annealed to the target were chain extended, while all non-annealed extension probes were cleaved to the position of the thio bond. The extension probe was radiolabeled at the 5'end to monitor degradation. Complete degradation of the unannealed extension probe up to the sulfur bond was obtained in about 1 minute, while the remaining sequences were resistant to degradation up to 60 minutes. After the completion of extension / degradation, the reaction solution is again heat-treated at 95 ° C. for 2 minutes, which inactivates a specific DNA polymerase and denatures the newly formed stem-loop from the original target molecule. . Thermostable polymerase was added (Stratagene, San D
iego, CA Pfu, 10 units per 100 μl), the reaction is temperature cycled as follows: 72 ° C.
Minute (1 time), 90 ° C 30 seconds, 55 ° C 60 seconds, 72 ° C
5 minutes (10 times), 90 ° C 30 seconds, 55 ° C 60 seconds and 72 ° C 90 seconds (50 times). The solution for these reactions is 1
xTBE buffer (89 mM Tris-boric acid, 89 m
1.2% agarose (Seakem, FMC BioProducts) in M boric acid, 0.2 mM EDTA).
The amplification products were analyzed by gel electrophoresis and visualized by ethidium bromide staining. The results are shown in Figure 14, lane 1 showing the extension probe at 0.5 micromolar concentration and lane 2 showing the extension probe at 1 micromolar concentration.

Example 4 PCR Amplification Using Thiophosphate Primer PCR amplification was 10 mM Tris-Cl, pH 8.3,
50 mM KCl, 1.5 mM MgCl2, 0.01
% Gelatin and 200 micromolar deoxynucleotide triphosphates (dNTPs) in a 100 microliter reaction system at 10 atmol single chain M13m
p19 (target DNA, Bethesda Research
Laboratories (BRL)). 1.0 micromolar primer 3 (5'-
GTT GAA ATT AAA CCA TCT C
AA GCC C-3 '(SEQ ID NO: 11) and primer 4 (5'-CAT AGT TAG CGT AA
C GAT CTA AAG TTT TGT C-
3 '(SEQ ID NO: 12)), the reaction system
Heat treated at 5 ° C for 5 minutes and cooled to room temperature for 15 minutes. Taq DNA Polymerase (Stratagen)
e) is added (5 units) and the temperature is cycled as follows: 90 ° C 30 seconds, 55 ° C 60 seconds, 72 ° C 90.
Seconds. This temperature circulation is repeated 30 times. The expected size of the amplification product is 880 base pairs. The solution (5 microliters) taken after 0, 10, 20 and 30 cycles from the PCR reaction is electrophoresed on a 1% agarose gel and stained with ethidium bromide. Based on the mobility of molecular weight standards, a single band of approximately 880 base pairs appears at 10, 20 and 30 cycles of PCR.

The target concentration is reduced to 10 attomoles per 100 microliter reaction and amplified using PCR. The individual reaction components followed the previously described parameters as well as the time and temperature cycling parameters. The reaction is heat treated at 95 ° C for 5 minutes and cooled to room temperature for 30 minutes. The solution (5 microliters) taken after 0, 15 and 30 cycles is electrophoresed on a 0.8% agarose gel and stained as described. About 8
A single band of 80 base pairs appears after 30 cycles of PCR.

The above example shows that a primer containing a 3'-terminal consisting of a nucleotide monophosphate having at least one phosphate-sulfur bond in place of the phosphorus-oxygen bond is effectively used for nucleic acid amplification. Shows.

The above discussion contains some theory of the mechanisms involved in the invention. These theories do not limit the invention, as it has been shown that the invention achieves the results described.

The above description and examples fully disclose the invention, including its preferred embodiments. Modifications of the methods described which are obvious to those skilled in the art such as molecular biology and related sciences are included in the interpretation of the following claims.

[Brief description of drawings]

FIG. 1 is a schematic view showing an embodiment of the method of the present invention.

FIG. 2 is a schematic view showing an embodiment of the method of the present invention.

FIG. 3 is a schematic view showing an embodiment of the method of the present invention.

FIG. 4 is a schematic view showing an embodiment of the method of the present invention.

FIG. 5 is a schematic view showing an embodiment of the method of the present invention.

FIG. 6 shows the use of the method of the invention in single primer amplification.

FIG. 7 is a schematic view showing an embodiment of the method of the present invention.

FIG. 8 is a schematic view showing an embodiment of the method of the present invention.

FIG. 9 is a schematic view showing a specific example of the second aspect of the present invention.

FIG. 10 is a schematic view showing a specific example of the second aspect of the present invention.

FIG. 11 is a schematic view showing a specific example of the second aspect of the present invention.

FIG. 12 is a photograph of an agarose gel (chromatography) of the obtained reaction product.

FIG. 13 is a photograph of an agarose gel (chromatography) of the obtained reaction product.

FIG. 14 is a photograph of an agarose gel (chromatography) of the obtained reaction product.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Karen M. Hahnenberger Portal Plaza, Cupertino, Calif., USA 19940 (72) Inventor Samuel Rose Inventor, Mountbury, California, USA 1008 (72) Inventor, Martin Becker, Palo Alt, Calif., USA 3481 (72) Invention Person Edwin F. Ullman Selby Lane, Atherton, California 135 (72) Inventor Glenn Eich. McGol, Mountain View, California, United States, California Street 2065

Claims (22)

[Claims] 1. From the extender probe and the single-stranded target polynucleotide sequence, a sequence identical to the target polynucleotide sequence without an unmodified extender probe is present at its 3'end at the 5'end of the target sequence. A method for forming a polynucleotide bound to a polynucleotide sequence complementary to a nucleotide sequence, comprising: (a) 5'of the single-stranded target polynucleotide sequence at the 3'end of the single-stranded target polynucleotide sequence. Hybridizing the 3'end of the extender probe containing a sequence substantially identical to sequence S2 at the end, (b) extending the extender probe along the single-stranded target polynucleotide sequence, (c) Modifying the 3'end of the extension probe that does not hybridize to the single-stranded target polynucleotide; Hybridized primers having sequence S2 at the 3 'end of the extended extender probe, and then (e) a method of the above primers to extend along the extended extender probe 2. The method of claim 1 wherein the modification comprises means for extending or degrading the 3'end of the extender probe that does not hybridize to the single stranded target polynucleotide sequence. 3. A polynucleotide (PN) having two non-adjacent and mutually complementary segments from an extended probe.
D), which is substantially free of the extension probe
A method for producing D, comprising: (a) two non-adjacent non-complementary nucleotide sequences S1
And S2, S2 is 5'of S1 and is at least 10
A polynucleotide having a nucleotide length, (b) an extension probe consisting of two deoxynucleotide sequences, the sequence at the 3'end of the extension probe, EP1
Is an extender probe hybridizable to S1 and the other deoxynucleotide sequence, EP2 is homologous to S2, and (c) means for chemically modifying the 3'end of the extender probe that does not hybridize to the polynucleotide. And the extension probe is extended along the polynucleotide, wherein the 3 ′ end of the extension probe that does not hybridize to the polynucleotide is modified. 4. The method according to claim 3, wherein the means comprises the nucleotide sequence EP3 capable of hybridizing with EP1. 5. The method of claim 3 wherein the means comprises the nucleotide sequence EP3 in the extension probe, which EP3 is hybridizable with EP1. 6. The method according to claim 3, wherein the means is a means for degrading the 3'end of the extension probe. 7. The method according to claim 6, wherein the means comprises an enzyme having 3'-endonuclease activity. 8. The method of claim 6 wherein the extension probe is degraded to provide a polydeoxynucleotide primer capable of hybridizing to a nucleotide sequence complementary to S2 at least at its 3'end. 9. A polydeoxynucleotide primer capable of hybridizing to a nucleotide sequence complementary to S2 at least at its 3 ′ end during the combination under the following conditions, which condition is (1) extension. The extended probe becomes a single strand, and (2) the polydeoxynucleotide primer hybridizes with the extended probe and extends along it to form a double strand consisting of the extended primer, (3) the extended primer dissociates from the duplex, and (4) the primer hybridizes with the extended primer and extends along it to form a duplex of the extended primer. The method according to claim 3, which is to be formed. 10. The method according to claim 9, wherein steps (3) and (4) are repeated. 11. S2 is 5'of S1 and is defined in "claim 3" for replicating a target polynucleotide sequence having noncontiguous non-complementary nucleotide sequences S1 and S2, each of at least 10 nucleotides in length. The method comprises: (A) some of the extender probes hybridize to the target polynucleotide sequence and extend along the target polynucleotide sequence (extended E
P) forms a double strand, and (B) an extension probe that does not hybridize to the target nucleotide sequence is extended or degraded at its 3'end, and (C) the extended EP is the same as in 2 above.
Dissociated from the strand, (D) the primer hybridizes to and extends along with the extended EP,
Forming a double strand consisting of the extension primer, (E) the extension primer is dissociated from the double strand, and (F)
The primer hybridizes with the extension primer and is extended along the extension primer to form a double strand consisting of the extension primer, and under the condition that steps (E) and (F) are repeated, (1) the target polynucleotide Array, (2)
The sequence EP1 at the 3'end of the extender probe is hybridizable with S1 and the other deoxynucleotide sequence EP2.
Is an extension probe having two deoxynucleotide sequences homologous to S2, (3) means for extending or degrading the 3'end of the extension probe that does not hybridize with the target polynucleotide sequence, (4) direct primer The polydeoxynucleotide primer provided, or produced in situ, with a 3'terminal sequence S2, (5) a DNA polymerase, and (6) a deoxynucleoside triphosphate, either simultaneously or in whole or in part. A method comprising the steps of sequentially providing the parts in combination. 12. The method of claim 11, wherein the polydeoxynucleotide primer is provided by the degradation of an extension probe and the phosphate closest to the 3'end of the primer is replaced with phosphorothioate. 13. A method according to claim 3 for detecting the presence of a polynucleotide, comprising: (a) treating a medium suspected of containing the polynucleotide (if present); Forming from the polynucleotide a single-stranded target polynucleotide sequence having two non-contiguous non-complementary nucleotide sequences S1 and S2, where S2 is 5'of S1 and is at least 10 nucleotides in length; b) In the above medium, (1) 3'of the extension probe
The terminal sequence (EP1) is hybridizable with S1,
The other deoxynucleotide sequence (EP2) is essentially S
An extension probe having two deoxynucleotide sequences identical to 2, (2) a nucleotide sequence (NS) having a portion capable of hybridizing with EP1, which is another molecule or portion of the extension probe. NS, (3) a polydeoxynucleotide primer capable of hybridizing with a nucleotide sequence complementary to S2, (4) deoxynucleoside triphosphate,
(5) and the DNA template-dependent polydeoxynucleotide polymerase, (A) the extension probe hybridizes with the target polynucleotide sequence, and is extended along the target polynucleotide sequence (extended extension probe). An extension probe that forms a double strand and does not hybridize to (B) the target polynucleotide sequence is N
Extended along S, (C) the extended extension probe is dissociated from the double strand, and (D) the primer hybridizes with and is extended along with the extended extension probe. Forming a double strand consisting of a primer, (E) the extension primer is dissociated from the double strand, and (F) the primer hybridizes with and extends along the extension primer to the extension primer. Forming a double strand and combining steps (E) and (F) under repeated conditions (where steps (a) and (b) are carried out simultaneously or wholly or partially sequentially) And (c) a step of examining the presence of the extension primer. 14. A method of forming a polynucleotide sequence identical to a single-stranded target polynucleotide sequence, the method comprising:
(A) Single-stranded target polynucleotide sequence in a medium, DN
A combination of one or more enzymes having A polymerase activity and 3'-exonuclease activity, and an extension probe, wherein the extension probe is at the 3'end and the sequence S1 at the 3'end of the single-stranded target polynucleotide sequence. EP1 which is complementary to and the sequence S at the 5'end of the above target sequence
2), wherein the extension probe contains at least one phosphorothioate within the EP2 sequence), and (b) treating the medium,
The extension probe is hybridized to the single-stranded target polynucleotide, the extension probe is extended along the single-stranded target polynucleotide sequence, and 3 of the extension probes that do not hybridize to the single-stranded target polynucleotide sequence. EP2 is added to the 3'end by degrading the'end.
A method comprising forming a primer having a sequence, hybridizing the primer to an extended extension probe, and extending the primer along the extended extension probe. 15. A sequence (EP1) which is hybridizable with the first sequence in the target polynucleotide sequence is designated as 3 '.
An extension probe having a sequence (EP2) at the end that is substantially identical to the second sequence of the target polynucleotide sequence (wherein the second sequence in the target polynucleotide sequence is 5'of the first sequence). Non-contiguous with the first sequence), a nucleotide sequence (NS) having a portion capable of hybridizing with EP1 (where NS is a molecule or part thereof different from the extension probe), and a polydeoxynucleotide When there is no other means for forming a primer, a polydeoxynucleotide primer capable of hybridizing to a sequence complementary to the second sequence is added,
A kit comprising comprising in a packaged combination. 16. A kit comprising an extension probe having at least one phosphorothioate diester and a 3′-exonuclease capable of hydrolyzing the extension probe. 17. A method according to claim 3 for detecting the presence of a polynucleotide, comprising: (a) treating a medium suspected of containing the polynucleotide (if present); Forming from the polynucleotide a single-stranded target polynucleotide sequence having two non-contiguous non-complementary nucleotide sequences S1 and S2, where S2 is 5'of S1 and is at least 10 nucleotides in length; b) In the above medium, (1) 3'of the extension probe
The terminal sequence (EP1) is hybridizable with S1,
The other deoxynucleotide sequence (EP2) is essentially S
An extender probe consisting of two deoxynucleotide sequences identical to 2 (wherein the extender probe is at least 1
Three phosphorothioate diesters, which can be degraded to form a polydeoxynucleotide primer capable of hybridizing to a nucleotide sequence complementary to S2), (3) deoxynucleoside triphosphates,
(4) and the DNA template-dependent polydeoxynucleotide polymerase, (A) the extension probe hybridizes with the target polynucleotide sequence and is extended along the target polynucleotide sequence to form a double strand, ( B) an extension probe that does not hybridize to the target nucleotide sequence is degraded to form the polydeoxynucleotide primer, (C) the extended extension probe is dissociated from the double strand, and (D) the primer is Hybridized with and extended along the extended extension probe, (E) the extension primer is dissociated from the double strand, and (F) the primer hybridizes with and extends along the extension primer. To form a double strand consisting of the extended primer,
Steps (E) and (F) in combination under repeated conditions (where steps (a) and (b) are carried out simultaneously or wholly or partly sequentially), and (c) the above extension. A method comprising the step of examining the presence of a primer. 18. A method of forming a polynucleotide sequence complementary to a single-stranded target polynucleotide sequence ("target sequence"), comprising: (a) a nucleotide monophosphate (herein) at the 3'end of the target sequence. At least one of which is sulfur in place of phosphate oxygen) and has a 3'end having a 3'end, and (b) the polynucleotide primer is extended along a target sequence, and (c) the target. A method comprising dissociating the extended polynucleotide primer from a sequence. 19. The method of claim 18, wherein there is sulfur in place of 1 to 5 phosphate oxygens. 20. A “claim” for forming a polynucleotide sequence having a sequence identical to a single-stranded target polynucleotide sequence (“target sequence”) except that it has a phosphorus-sulfur bond instead of a phosphorus-oxygen bond. Item 18 ”, wherein (a) the 3 ′ end of the target sequence is hybridized with a first polynucleotide primer containing at least one phosphorus-sulfur bond (“ first primer ”), (B) extending the first primer along the target sequence, (c) dissociating the extended first primer from the target sequence, and (d) the extended first primer.
To the 3'end of the first primer, a second polynucleotide primer that is the same as or different from the first primer ("second primer"), and (e) the second primer Extending along the primer to form an extended second primer (where the first primer and the second primer are the same, the extended first primer is the template of the first primer, If the first primer and the second primer are different, the extended first primer is the template of the second primer, and the extended second primer is the template of the first primer) How to become. 21. The method of claim 20, wherein the first primer and the second primer are the same. 22. The “claim 20” or “claim” wherein the conditions for repeated cycling are selected such that the extended primer is dissociated from its template and then the primer is extended along the extended primer. Item 2
The method described in 1 ”.